Tri-hybrid melanoma antigen

ABSTRACT

The present invention relates to novel tri-hybrid melanoma antigens, including antigenic fragments or derivatives thereof, of a tyrosinase (TYR) antigen, a tyrosinase-related protein 1 (TRP-1) antigen, and a tyrosinase-related protein 2 (TRP-2) antigen and nucleic acids encoding them. The novel tri-hybrid melanoma antigens of the present invention are useful in the diagnosis, treatment and prevention of human neoplasms, including malignant tumors, such as carcinomas, sarcomas, leukemia, and lymphomas, and pre-malignant lesions, such as adenomas and dysplastic lesions.

FIELD OF THE INVENTION

[0001] The present invention relates to novel tri-hybrid melanoma antigens, including antigenic fragments or derivatives thereof, of a tyrosinase (TYR) antigen, a tyrosinase related protein-1 (TRP-1) antigen, and a tyrosinase related protein-2 (TRP-2) antigen and nucleic acids encoding them, which are useful in the prevention and treatment of human neoplasms.

BACKGROUND OF THE INVENTION

[0002] Cancer cells are not static in nature, but are changing constantly. To escape immunosurveillance and multiply, cancer cells have developed a number of different mechanisms, include the following: decreasing expression in MHC molecules (see Ferrone et al., Immunol. Today, 16:487-494 (1995)); deficiencies in antigen processing (see Kamarashev et al., Int. J. Cancer, 95:23-28 (2001)); secretion of immune suppressing cytokines (see Spellman et al., Surg Oncol., 5(5-6):237-44 (1996)); and loss of tumor antigen expression (see Ohmacht et al., J. Cell Physiol., 182: 332-338 (2000)).

[0003] With respect to the loss of tumor antigen expression, a number of melanoma-specific antigens have been identified in recent years. One major group of such antigens, which are recognized by the immune system, consists of melanocyte differentiation antigens, such as tyrosinase, TRP-1 (also designated gp75), TRP-2, gp100 and MART-1/Melan-A. All of these antigens are present in melanosomes.

[0004] Tyrosinase, TRP-1, and TRP-2, are melanocyte differentiation antigens, located in the melanosomes of melanocytes and melanomas, and involved in melanin synthesis. See Kawakami et al., Immunol. Res., 16: 313-339 (1997); Kawakami et al., J. Immunother., 21: 237-246 (1998). Human tyrosinase, a 529 amino acid melanosomal membrane protein, has tyrosine hydroxylase, DOPA oxidase and DHI activity, and is the principal enzyme involved in melanin synthesis. Human TRP-1 consists of 537 amino acids and has DHI-2-carboxylic acid oxidase activity. Human TRP-2 is a 519 amino acid melanosomal enzyme with DOPAchrome tautomerase activity. Antibodies and T cells to these antigens have been identified in melanoma patients. See Houghton et al., Ann. N.Y. Acad. Sci., 690: 59-68 (1993); Brichard et al., J. Exp. Med., 178: 489-495 (1993); Kang et al., J. Immunol., 155: 1443-1348 (1995); Wang et al., J. Exp. Med., 181: 799-804 (1995); Wang et al., J. Exp. Med., 184: 2207-2216 (1996). However, it remains unclear how tolerance to these differentiation antigens is broken in cancerous state.

[0005] Animal models of cancer vaccines have demonstrated the feasibility of melanoma vaccines using the tyrosinase family of proteins. Vaccination of mice with recombinant vaccinia viruses expressing mTRP-1 (see Overwijk et al., Proc. Nat'l Acad. Sci. USA., 96: 2982-2987 (1999)), or naked DNA encoding hTRP-1 (see Weber et al., J. Clin. Invest., 102: 1258-1264 (1998)), or insect cells containing mTRP-1 (see Naftger et al., Proc. Nat'l Acad. Sci. USA., 93: 14809-14814 (1996)) have demonstrated the ability to break tolerance to TRP-1 and have shown induction of strong antitumor activities, which were dependent on TRP-1 specific antibodies and CD4 T cells. Immune tolerance to TRP-2 also has also been broken with naked DNA encoding human TRP-2, following induction of CD8 T cells (see Browne et al., J. Exp. Med., 190: 1717-1722 (1999)).

[0006] Many of the clinical vaccine trials targeting the tyrosinase family of proteins are underway to treat melanoma. It has been reported that melanoma cells can lose the expression of TRP-1, TRP-2, or tyrosinase. See Id., Marincola et al., Adv. Immunol., 74: 181-273 (2000). However, the likelihood that a melanoma will lose all three antigens at same times is low; therefore, a chimeric protein containing all three of these melanoma antigens will have great advantage.

[0007] Most of the vaccine approaches are based on defined peptide epitopes, plasmid DNA, and recombinant viruses, each of which suffers from various disadvantages. The identification of HLA-binding epitopes and HLA types of each patient are needed for peptide epitope-based vaccine. Plasmid DNAs in general are week immunogens. Recombinant viruses generate neutralizing antibodies, which limit the administration of vaccine. As such, use of a recombinant protein vaccine is an attractive alternative approach for a cancer vaccine.

[0008] There are several advantages to use of a recombinant protein vaccine, including the fact that the vaccine can be administered repeatedly, it can induce a wise spectrum of immune responses, including production of antibodies, cytotoxic T-lymphocytes (CTLs) (with appropriate adjuvants), and CD4+ T cells (important in maintaining tumor immunity). Moreover, a protein vaccine can contain all possible epitopes of the antigen.

SUMMARY OF THE INVENTION

[0009] The present invention is directed to tri-hybrid melanoma antigens, and isolated DNAs encoding such antigens, comprising tyrosinase or a fragment thereof, TRP-1 or a fragment thereof, or TRP-2 or a fragment thereof. The present invention is also directed to compositions for inhibiting melanosomal activity or tumor growth comprising such tri-hybrid melanoma antigens and isolated DNAs. In addition, the present invention is directed to methods of eliciting an immune response against a melanosomal antigen, methods of treating a tumor, or methods of vaccination using such tri-hybrid melanoma antigens and isolated DNAs.

DESCRIPTION OF THE FIGURES

[0010]FIG. 1 shows construction of a tri-hybrid melanoma antigen, hTRPx3, containing human TRP-1, TRP-2, and tyrosinase. A tri-hybrid melanoma antigen DNA fragment was generated with primers and the resulting fragments were mixed and fused following 10 PCR cycles. The final chimeric DNA was generated with primers htyrF-1 (SEQ ID NO:1) and htyrF-6 (SEQ ID NO:6) and then cloned into bacterial expression vector pET28a(+).

[0011]FIG. 2 shows purification and characterization of a recombinant tri-hybrid melanoma antigen, hTRPx3 protein, by SDS-PAGE and Western blotting. In FIG. 2A, an SDS-PAGE gel shows the expression and purification of the hTRPx3 protein and in FIG. 2B, a Western blot shows that the purified hTRPx3 protein is recognized by antibodies specific for human TRP-1, TRP-2, and tyrosinase.

[0012]FIG. 3 graphically demonstrates the antibody responses in mice following immunization with a tri-hybrid melanoma antigen, hTRPx3 protein. In FIG. 3A, a graph shows that hTRPx3 protein immunization induced antibodies against TRP-1, TRP-2, and tyrosinase and hTRPx3. In FIG. 3B, a graph shows the isotypes of the antibodies specific to the hTRPx3 protein.

[0013]FIG. 4 graphically demonstrates that immunization of mice with a tri-hybrid melanoma antigen, hTRPx3, resulted in production of IFNγ releasing T cells specific for TRP-1, TRP-2, and tyrosinase.

[0014]FIG. 5 shows induction of a T cell immune response following immunization with a tri-hybrid melanoma antigen, hTRPx3 protein. In FIG. 5A, an ELISPOT blot shows induction of IFNγ-producing T cells, and in FIG. 5B, this IFNγ-producing T cell induction is represented graphically.

[0015]FIG. 6 shows that immunization with a tri-hybrid melanoma antigen, hTPRx3 protein, was useful in treating tumors in mammals. In FIG. 6A, the number of lung surface metastases following immunization is represented graphically. In FIG. 6B, the number of lung surface metastases following immunization of MHC class I knock-out mice, MHC class II knock-out mice, and FcRγ knock-out mice is represented graphically.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention relates to tri-hybrid melanoma antigens of a tyrosinase antigen (U.S. Pat. No. 4,898,814), a TRP-1 antigen (WO 91/14775) and a TRP-2 antigen (U.S. Pat. No. 5,831,016), including antigenic fragments and derivatives thereof. Derivatives include, for example, antigens that are mutational or allelic variants. The antigens can be of human or, more generally, of mammalian origin, and the components of the tri-hybrid melanoma antigen can be from different sources (e.g., homologous antigens from different species). Moreover, each component (i.e., antigen or antigenic fragment) can be a hybrid combining sequences from more than one source. The invention provides a tri-hybrid melanoma antigen that is more effective for immunization against melanoma than any single antigen from which it is derived.

[0017] “Antigenic fragment,” as the term is used herein, means any antigenic segment of a protein or gene, usually having at least 5 or 6 amino acids in the case of a protein fragment and at least 15-18 nucleotides in the case of a gene. Thus, a fragment generally encompasses a segment of a protein, or the nucleotide sequence that encodes it, which comprises at least one B cell or T cell epitope.

[0018] Tyrosinase, TRP-1 (also known as gp75) and TRP-2 are expressed primarily in melanomas, normal melanocytes, and in the retina. The three proteins are related in sequence, sharing (pairwise) greater than 40% amino acid sequence identity and greater than 50% amino acid sequence similarity, and have in common N-terminal signal peptides and a C-terminal sequence involved in intracellular retention and sorting to melanosomes along the endosomal/lysosomal pathway. Moreover, these three members of the tyrosinase family of proteins are highly conserved among species.

[0019] The invention further relates to homologs of human tyrosinase, TRP-1 and TRP-2, that can be used to break tolerance in humans to the human proteins. Table 1 provides two examples of such homologs for each of these three tyrosinase family proteins. The value for percent identity of the homolog to the human protein is calculated over the entire length of the protein. Homologs that can be used according to the invention have at least 60% identity to the corresponding protein of the species in which tolerance is to be broken. Preferred homologs have at least 70% identity. More preferred homologs have at least 80% identity. It is noted that where less than a complete amino acid sequence is to be incorporated into a tri-hybrid melanoma antigen, percent identity is calculated only over the length of the protein fragment that is incorporated.

[0020] Nucleotide sequences of mRNAs encoding human tyrosinase, TRP-1/gp75 and TRP-2 and the sequences of the proteins themselves are publicly available from GenBank (National Center for Biotechnology Information, National Library of Medicine, Bethesda, Md.), as are homologous sequences from other species. Nucleotide and amino acid sequences referred to herein correspond to GenBank accession numbers as given in Table 1. The sequences give therein are meant as examples only, and do not limit the scope of the invention. TABLE 1 Nucleotide Amino Acid Percent Identity Source Sequence Sequence to Human Protein Tyrosinase Human NM_000372 NP_000363 — Mouse NM_011661 NP_035791 86 Rabbit AF210660 AAF43895 89 TRP-1 Human NM_000550 NP_000541 — Mouse MMTYRR TYR1_MOUSE 85 Goat AF136926 AAD34802 88 TRP-2 Human NM_001922 NP_001913 — Mouse NM_010024 NP_034154 83 Chicken AF023471 AAC63434 69

[0021] A preferred tri-hybrid melanoma antigen of the present invention comprises the soluble portion of each of tyrosinase, TRP-1 and TRP-2. For example, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11 provide examples of DNA sequences encoding a tri-hybrid melanoma antigen in the context of the present invention. The corresponding protein sequence for the tri-hybrid melanoma antigen is set forth in SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12. Accordingly, in one embodiment, an isolated DNA encoding the tri-hybrid melanoma antigen comprises SEQ ID NO:7 or a fragment thereof, SEQ ID NO:9 or a fragment thereof, or SEQ ID NO:11 or a fragment thereof and the tri-hybrid melanoma antigen itself comprises SEQ ID NO:8 or a fragment thereof, SEQ ID NO:10 or a fragment thereof, or SEQ ID NO:12 or a fragment thereof.

[0022] An example of a more preferred tri-hybrid melanoma antigen comprises the sequence from about amino acid residue 25 to about amino acid residue 477 of human TRP-1 or a homolog thereof, the sequence from about amino acid residue 24 to about amino acid residue 472 of human TRP-2 or a homolog thereof, and the sequence from about amino acid residue 19 to about amino acid residue 476 of human tyrosinase or a homolog thereof. The individual fragments can be linked in any order and the tri-hybrid melanoma antigen can further comprise glycine residues by which the fragments are linked.

[0023] Thus, a particularly preferred tri-hybrid melanoma antigen is represented by SEQ ID NO:8 from about amino acid residue number 25 to about amino acid residue number 1392 (containing glycine linkages) and SEQ ID NO:12 from about amino acid residue number 25 to about amino acid residue number 1384 (without glycine linkages). It will be apparent to one of ordinary skill in the art that there can be some variation in the extent of the protein fragments of which the tri-hybrid melanoma antigen is comprised, which will have little or no effect on its immunogenic properties.

[0024] Where non-human mammalian proteins, mutational variants, hybrids, fragments, or derivatives are used, they can be selected such that they possess desirable properties such as increased immunogenicity, decreased side effects, and increased half-life. For example, fragments of the individual antigens can be selected for incorporation into a tri-hybrid melanoma antigen of the invention based on the presence of known or postulated B cell or T cell epitopes. As another example, mutational variants that exhibit improved binding to MHC molecules can be selected.

[0025] Production of large quantities of tri-hybrid melanoma antigens can be accomplished by methods known in the art. For example, large amounts of tri-hybrid melanoma antigens can readily be synthesized in vitro. As another example, nucleic acid encoding tri-hybrid melanoma antigens can be transfected into bacterial, insect, or mammalian cells using appropriate vectors and methods as known in the art. Accordingly, the present invention encompasses cloning or expression vectors comprising a DNA encoding a tri-hybrid melanoma antigen and host cells comprising such cloning or expression vectors.

[0026] Where needed or desired for expression of the tri-hybrid melanoma antigen in a given cell type, the first protein fragment of the tri-hybrid melanoma antigen will be preceded by a signal peptide, which can be the signal peptide that is native to the first protein fragment in the tri-hybrid melanoma antigen, or can be a signal peptide derived from another source. For example, a signal sequence signaling for secretion is useful where it is desired to provide for secretion of a tri-hybrid melanoma antigen into external mileau of a cell. For a review of secretion and signal peptide function, see, for example, Pugsley, Curr. Opin. Cell Biol., 2:609-616 (1990). Algorithms and computer implementations for secretory signal sequence prediction are available. See, e.g., von Heijne et al., Nucleic Acids Res., 14:4683-4690 (1986); Nielsen et al., Protein Eng., 10:1-6 (1997).

[0027] For expression in bacterial cells, a bacterial signal sequence can be preferred. Alternatively, a signal sequence is not necessary to express the tri-hybrid melanoma antigen in a bacterial host in inclusion bodies. For example, a tri-hybrid melanoma antigen of the invention can comprise the amino acid sequence represented by SEQ ID NO:8 from about amino acid residue number 25 to about amino acid residue number 1392. Such a tri-hybrid melanoma antigen can be expressed with an N-terminal methionine residue, depending on the nature of the host bacteria. The methionine can be cleaved in vivo in the bacterial host cell or remain intact. Such a tri-hybrid melanoma antigen can be expressed in E. coli and obtained from inclusion bodies by methods well known in the art.

[0028] To assist in purification, tri-hybrid proteins of the invention can be expressed with fused “tag” sequences. For example, the tri-hybrid melanoma antigen encoding DNA sequence can be cloned in an expression vector that provides for production of the tri-hybrid melanoma antigen linked to an N-terminal His tag sequence. The His tag allows purification by metal chelation chromatography. In certain embodiments, the His tag can be cleaved from the tri-hybrid melanoma antigen after purification. Alternatively, tag sequences that provide for affinity purification can be used. In a preferred embodiment, a tri-hybrid melanoma antigen is expressed from pET-28a(+), purified by metal chelation chromatography, and the His tag removed by specific proteolysis at the thrombin cleavage site.

[0029] The invention provides novel tri-hybrid melanoma antigens that are particularly useful as vaccines for inducing immune responses effective for treating, inhibiting, and preventing cancers and precancers. Of particular interest are tri-hybrid melanoma antigens that induce anti-tumor immune responses in patients with melanoma. Accordingly, the present tri-hybrid melanoma antigens can be administered for prophylactic and/or therapeutic treatments of various conditions. Treatment, in the context of the present invention, is intended to encompass inhibiting, slowing, or reversing the progress of the underlying condition, ameliorating clinical symptoms of a condition or preventing the appearance of clinical symptoms of the condition.

[0030] The term “melanoma” includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocyte related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma, invasive melanoma and familial atypical mole and melanoma (FAM-M) syndrome.

[0031] Many methods suitable for administering the tri-hybrid melanoma antigen are known in the art. For example, the tri-hybrid melanoma antigen can be administered in soluble form. As another example, autologous mammalian cells capable of expressing the tri-hybrid melanoma antigen can be administered. As another example, virus having tri-hybrid melanoma antigen on its surface can be administered. As yet another example, virus carrying nucleic acid encoding the tri-hybrid melanoma antigen can be administered. As still another example, naked DNA or other nucleic acid encoding the tri-hybrid melanoma antigen can be administered.

[0032] Tri-hybrid melanoma antigens can be administered alone, combined with adjuvants, linked to helper (carrier) peptides, proteins, lipids or liposomes, or pulsed onto purified antigen presenting cells (APCs) and the antigen presenting cells used for immunization. Adjuvants for use in immunization and other treatment methods include, for example, RIBI Detox (Ribi Immunochemical), QS2 1, CRIS-2 1, alum, BCG and incomplete Freund's adjuvant. For test animals, adjuvants further include complete Freund's adjuvant and others commonly used in the art. Tri-hybrid melanoma antigens can be also be complexed with heat shock binding proteins. APCs are generally eukaryotic cells with major histocompatibility complex (MHC), either class I or class II, gene products at their cell surface. Some examples of APCs that can be used in the present invention include DC, as well as macrophages, preferably MHC class II positive macrophages, monocytes, preferably MHC class II positive monocytes, and lymphocytes. See generally U.S. Pat. No. 5,597,563. It should be appreciated that such administration can be carried out before, simultaneously with, or after administration of the novel tri-hybrid melanoma antigens.

[0033] Tri-hybrid melanoma antigens can be administered as DNA vaccines. DNA vaccines can comprise “naked” DNA encoding tri-hybrid melanoma antigens. Preferably, the tri-hybrid melanoma antigen-encoding DNA is taken up by host cells and expressed polypeptides are efficiently presented to the immune system. For example, naked DNA can be injected intradermally or intramuscularly or linked to lipids. Preferably, vaccines comprising tri-hybrid melanoma antigen injected directly into muscle or into the skin raise both cellular and humoral immune reactions to encoded antigens. See, for example, U.S. Pat. No. 5,831,016, Gregersen, Naturwissenschaften, 88(12):504-13 (December 2001), and Wang et al., Expert Opin. Biol. Ther., 1(2):277-90 (March 2001) for methods of preparation and use of DNA vaccines. Vaccines can comprise non-viable DNA vectors comprising DNA encoding tri-hybrid melanoma antigen of the present invention. Non-viable DNA vectors have the advantage of ease of preparation and safety of administration. DNA sequences encoding tri-hybrid melanoma antigens of the present invention can be administered using a gene gun in amounts to elicit a cellular response against a cancer cell. Nanogram quantities can be useful for such purposes.

[0034] DNA encoding tri-hybrid melanoma antigens can also be expressed by bacteria or from recombinant viruses upon infection of host cells of the patient. Such bacterial or viral vectors can be designed to also express co-immunostimulatory molecules which enhance an immune response. Co-immunostimulatory molecules include, for example, IL-2, IL-6, IL-10, IL-12, and γ-interferon (IFN-γ). Co-immunostimulatory molecules can be selected so as to favor humoral immune responses or cytotoxic immune responses.

[0035] DNA encoding tri-hybrid melanoma antigens of the present invention can also be used to create genetically modified immune cells capable of recognizing human tumor antigens. Such genetically modified immune cells can be particularly effective in, for example, mediating the regression of cancer in selected patients with metastatic melanoma. Techniques by which human lymphocytes are sensitized in vitro to tumor antigen peptides presented on antigen presenting cells are known in the art. By repetitive in vitro stimulation cells can be derived with a far greater capacity to recognize and respond to human tumor antigens. Thus by repeated in vitro sensitization with the tumor antigen of the present invention, lymphocytes can be derived with increased potency. The cells to be sensitized can be obtained from the subject to be treated or can be MHC matched cells from other sources. Adoptive transfer of these cells into the subject to be treated can result in increased effectiveness in mediating tumor regression in vivo.

[0036] Tri-hybrid tumor antigens can be administered via one or more of several routes including, but not limited to, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal, intrapleural, intrauterine, rectal, vaginal, topical, intratumor and the like.

[0037] Administration can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be by nasal sprays, for example, or suppositories. For oral administration, the tumor antigen, cancer peptides or variants thereof are formulated into conventional oral administration forms such as capsules, tablets and tonics.

[0038] It is understood that the tri-hybrid melanoma antigens of the present invention, where used in an animal for the purpose of prophylaxis or treatment, can be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins. The compositions of the injection can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal.

[0039] The compositions of this invention can be in a variety of forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions, dispersions or suspensions, liposomes, suppositories, injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application.

[0040] Such compositions of the present invention are prepared in a manner well known in the pharmaceutical art. In making the composition the active ingredient will usually be mixed with a carrier, or diluted by a carrier and/or enclosed within a carrier which can, for example, be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, excipient or medium for the active ingredient. Thus, the composition can be in the form of tablets, lozenges, sachets, cachets, elixirs, suspensions, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, injection solutions, suspensions, sterile packaged powders and as a topical patch.

[0041] It should be appreciated that the immunogen of the present invention can be administered to any suitable animal. For example, the animal is preferably a mammal, such as a rabbit, rat, or mouse. More preferably, the animal is a human.

[0042] The tri-hybrid tumor antigen according to the present invention is preferably provided in a therapeutically effective amount. Preferably, the dose is effective to prime, stimulate and/or cause the clonal expansion of cancer antigen specific B and T lymphocytes, which in turn are capable of preventing, inhibiting, or treating cancer in the recipient.

[0043] Therapeutically effective doses can be determined by those skilled in the relevant arts via clinical studies. Therapeutically effective doses can also be determined by in appropriate animal models, then extrapolated to humans using known techniques. For example, for systemic administration, the amount of compound administered per unit body weight determined in rats can easily be applied to humans.

[0044] Therapeutically effective doses will vary, depending on such factors as the weight and condition of the patient, the type of melanoma or other cancer to be treated, inhibited, or prevented, and the method of administration.

[0045] The dosage of tri-hybrid tumor antigen for a human can be at least about 1 pg per Kg of body weight. A range of from about 1 ng per Kg body weight to about 100 mg per Kg body weight is preferred. More preferably, the amount administered can be at least about 1 □g per Kg body weight to about 1 mg body weight.

[0046] The dose is administered at least once and can be provided as a bolus or a continuous administration. Multiple administrations of the dose over a period of several weeks to months can be preferable. Where multiple doses are provided, the dosage amount and formulation can be the same or can differ among doses.

[0047] Effective treatment of melanoma or other cancer or pre-malignant lesion can be measured by many parameters known in the art, including, but not limited to, the decrease of tumor burden, increase in humoral immune response, changes in serum tumor markers, and increase in cytotoxic or cell-mediated immune responses. As more specific examples, antibody levels and CTL levels can be measured. Still other examples of criteria that can be used to evaluate effective treatment include standard World Health Organization (WHO) criteria for tumor response. Effective prevention of melanoma can be measured by many parameters known in the art, including, but not limited to, the decrease of incidence in a treated population compared to an untreated population. Such criteria can be measured by methods known in the art.

[0048] Accordingly, the present invention can be used in vivo and in vitro for investigative, diagnostic, prophylactic, or treatment methods, which are well known in the art.

[0049] Of course, it is to be understood and expected that variations in the principles of the invention herein disclosed can be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention.

[0050] All references mentioned herein are incorporated in their entirety.

EXAMPLES

[0051] The examples that follow further illustrate the invention, but should not be construed to limit the scope in any way. Detailed descriptions of conventional methods, such as those employed in the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids, the introduction of plasmids into host cells, and the expression and determination thereof of genes and gene products can be obtained from numerous publications, including Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press.

[0052] General Methods

[0053] Cells and Animals

[0054] The mouse melanoma cell line B16BL6 was kindly provided by Dr. Isaiah Fidler (M. D. Anderson Cancer Center, Houston); B16BL6 expression of TRP-1, TRP-2, and tyrosinase was determined by RT-PCR and Western analysis. The EL4 cell line was obtained from American Type Culture Collection (Manassas, Va.). C57BL/6 and C57BL/6 with deficiencies in MHC class I, or MHC class II, or FcR were purchased from Jackson Laboratory (Bar Harbor, Me.). All cell lines were maintained in RPMI 1640 (Gibco BRL, Gaithersburg, Md.) with 10% heat-inactivated fetal bovine serum and without antibiotics and were routinely tested for Mycoplasma contamination and were negative.

[0055] All experiments and procedures were performed in accordance with the United States Department of Agriculture and Human Services, and NIH policies regarding the use of laboratory animals.

[0056] Generation of a DNA Fragment Containing hTRP-1, hTRP-2, and h-Tyrosinase

[0057] To generate a chimeric DNA fragment, three pairs of primers were designed. Primer pair one used hgp75 as template, primer pair two used hTRP-2 as template, and primer pair three used h-tyrosinase as template. Pair one, termed htyrF-1 and htyrF-2, generated a DNA fragment containing a soluble hgp75, four-glycines linker and the 5′ portion of hTRP-2 (htyrF-1 (SEQ ID NO:1): 5′ gccgaatcca tgagtgctcc taaactcctc 3′ and htyrF-2 (SEQ ID NO:2): 5′ cgtcatgcag actcggggga actgccctcc gccaccctca ggtacactaa actcccgact tgg 3′). Pair two, termed htyr-3 and htyr-4, cover the 3′ portion of hgp75, the soluble portion of hTRP-2, four glycines linker, and the 5′ portion of h-tyrosinase (htyrF-3 (SEQ ID NO:3): 5′ ccaagtcggg agtttagtgt acctgagggt ggcggagggc agttcccccg agtctgcatg acg 3′ and htyrF-4 (SEQ ID NO:4): 5′ ggagacacag gctctaggga aatgtccacc cccgccagtt gtgggccaac ctggagtttc 3′). Pair three, termed htyr-5 and htyr-6, PCR-cloned a fragment containing the 3′ portion of h-tyrosinase, the soluble portion of h-tyrosinase, and the stop codon (htyrF-5 (SEQ ID NO:5): 5′ gaaactccag gttggcccac aactggcggg ggtggacatt tccctagagc ctgtgtctcc 3′ and htyr-6 (SEQ ID NO:6): 5′ gcggcgctcg agctatgacc agatccgact cgcttg 3′). Each PCR reaction resulted in a 1.4 kb fragment (PCR product one). The resulting three PCR products were then mixed and underwent 10 cycle PCR reactions (PCR product two). Finally, chimeric DNA was generated with primers htyr-1 and htyr-6 (PCR product three). PCR product three was about 4.1 kb and digested with EcoR I and Xho I before cloning into pET28a(+) (Novogen, Madison, Wis.).

[0058] Expression and Purification of Recombinant hTRPx3

[0059]E. coli BL21 (Novogen, Madison, Wis.) transformed with pET28a(+) containing hTRPx3 cDNA were grown to a cell density of 0.6 (A₆₀₀ nm) at 37° C. in shake flasks. The lac promoter was induced by addition of IPTG to a final concentration of 1 mM, and cells were grown for an additional 4 hrs at 37° C. The cells were harvested by centrifugation, resuspended in PBS and disrupted with a Cell Disrupter (Constant Systems Ltd, Warwick, UK). The insoluble fraction of the E. coli homogenate, which contained recombinant inclusion bodies, was harvested by centrifugation (10,000 g, 4° C., 30 min). The pellets were dissolved in a solution containing 8M urea, 50 mM Tris, and 5 mM imidazole at pH 8.0. The recombinant hTRPx3 were affinity purified using Toyopearl His binding resin according to manufacturer's instruction (TosoHaas, Montgomeryville, Pa.). The recombinant human TRP-1, TRP-2, tyrosinase was expressed and purified in a similar manner.

[0060] Western blots were performed to ascertain that the recombinant hTRPx3 was recognized by antibodies specific for hTRP-1, hTRP-2, and h-tyrosinase. The purified hTRPx3 protein was run on 12% SDS-PAGE and transferred to PVDF membranes (Novex, San Diego, Calif.). The membranes were blocked overnight at 4° C. in 5% non-fat dry milk (blocking buffer) and probed with mouse sera specifically for individual antigens diluted 1:500 in PBS-0.2% Tween-20 at room temperature for 1 hr with gentle agitation. Blots were then washed with PBS-0.2%Tween-20 solution and incubated with peroxidase-conjugated goat anti-mouse IgG (Biosource, Camarillo, Calif.) diluted in PBS-0.2% Tween-20 (1:5000) for 1 hr. Blots were washed extensively as above and detected via ELC Western blotting reagents (Amersham Pharmacia Biotech, Little Chalfont, UK). Mouse sera specific for hTRP-1, hTRP-2, and h-tyrosinase were generated from mice immunized with individual proteins.

[0061] Enzyme-Linked Immunosorbent Assay (ELISA)

[0062] An indirect ELISA was developed to detect antibody responses. Purified human protein antigens, 50 ng in 50 μl PBS buffer, was added to each well of a 96-well Immunolon-2 plate (Dynex Technologies, Chantilly, Va.) and incubated overnight at 4° C. Plates were washed twice with PBS-0.2% Tween-20 and then incubated in blocking buffer (PBS containing 5% non-fat dry milk) at room temperature for 2 hrs. Mouse sera diluted at different concentrations in PBS-0.2% Tween-20 were added to plates and incubated at room temperature for 2 hrs. Wells were washed three times as above and then 100 μl peroxidase conjugated goat anti-mouse IgG (Biosource, Camarillo, Calif.) diluted 1:5000 in PBS-0.2% Tween-20 was added and incubated for 1 hr at room temperature. Wells then were washed three times and the peroxidase substrate TMB (KLP, Gaithersburg, Md.) was added. The absorbance at 450 nm was read on a kinetic microplate plate reader (Molecular Devices, Sunnyvale, Calif.).

[0063] IFNγ Release Assay

[0064] Splenocytes (4×10⁵) were harvested one week after the last immunization and cultured with 10 mg/ml recombinant proteins in a total volume of 2 ml of RPMI 1640 with 10% fetal bovine serum in a 24 well plate for 72 hrs. The supernatant were harvested and tested for IFNγ using ELISA kits (Research Diagnostics Inc., Flanders, N.J.).

[0065] ELISPOT Assay

[0066] To conduct ELISPOT assay, 1×10⁴ fresh isolated spleen cells from each vaccinated mice group were added to each well of 96 well plate along with 20 IU/ml IL-2. Cells were incubated at 37° C. for 24 hrs either with B16 or control EL4 cells. After culture, the plate was washed and then followed by incubation with 5 mg/ml biotinylated IFNγ antibody (clone XMG1.2, PharMingen) in 50 ml in PBS at 4° C. overnight. After washing six times, 1.25 mg/ml avidin-alkalinephosphatase (Sigma, St. Louis, Mo.) in 50 ml of PBS were added and incubated for 2 hrs at room temperature. After washing, spots were developed by adding 50 ml of 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium solution (Boehringer Mannheim, Indianapolis, Ind.) and incubated at room temperature for 1 hr. The spots were counted using a dissecting microscope.

[0067] Immunization and Tumor Protection Assay

[0068] One hundred micrograms of purified hTRPx3 was emulsified in 100 □l complete Freund's adjuvant (CFA) for each injection. C57BL/6 mice were vaccinated subcutaneously every 10 days for five times and bled one week after boosting. To compare different adjuvant, some mice were immunized with hTRPx3/BCG or hTRPx3/GMCSF intradermally. Unless stated, all results were from mice vaccinated with hTRPx3/CFA. For melanoma protection assay, mice were injected intravenously through the tail vein with 5×10⁴ B16BL6 cells 10 days after last immunization. Mice were sacrificed and lungs were removed 20 days after B16 melanoma challenge. The surface lung metastases were scored and counted under a dissecting microscope. Statistical analysis of surface lung metastases was performed using student t test.

Example 1

[0069] The present example demonstrates construction and production of a tri-hybrid melanoma antigen, recombinant hTRPx3 protein.

[0070] To generate a chimeric hTRPx3 molecule, a DNA fragment encoding human TRP-1, TRP-2, and tyrosinase were made using PCR primer pairs as shown above (FIG. 1). Each PCR resulted a 1.4 kb fragment (PCR product one). The resulting three PCR products were then mixed and underwent a 10-cycle PCR reaction (PCR product two). Finally, PCR product two was amplified with primers htyr-1 and htyr-6 (PCR product three). PCR product three was about 4.1 kb and was digested with EcoR I and Xho I before cloning into pET28 (Novogen, bacteria expression vector). The final product was confirmed by sequencing. The coding sequence of the cloned tri-hybrid nucleotide sequences was determined by standard techniques and is given by SEQ ID NO:7. The amino acid sequence of the translation product is given by SEQ ID NO:8.

[0071] Induction of BL21 E. coli cells containing plasmids encoding human hTRPx3 genes resulted in expression of 155-kDa-fusion proteins in insoluble forms (FIG. 2A). The supernatant fraction (i.e. the soluble protein) from cell lysates showed little amounts of hTRPx3 proteins in soluble form. No inhibition of cell growth during the induction was observed, indicating that human TRPx3 was not toxic when expressed in the fusion proteins. The level of expressed proteins could be increased if cells were induced at 37° C. due to the formation of inclusion bodies. The level of the expressed TRP1 protein was about 5 mg/L. The expressed hTRPx3 proteins were conformed by Western analysis using anti-sera specific for either TRP-1, or TRP-2 or tyrosinase (FIG. 2B).

Example 2

[0072] The present example demonstrates induced of a humoral immune response following immunization with a tri-hybrid melanoma antigen, hTRPx3 protein.

[0073] Protein immunization with hTRPx3 protein was performed on 10 mice per group. One week after last boost, serum samples were tested for their ability to react with the purified individual antigens on ELISA plates (FIG. 3A). A majority of immunized mice developed antibody responses against individual proteins and hTRPx3 (27/30).

[0074] To determine the subtypes of antibodies involved in these responses, antibody subtyping was performed on the ELISA plates (FIG. 3B). Briefly, the immune plates were coded with recombinant hTRPx3 and then incubated with sera from immunized mice. The bound antibodies were determined by second antibodies specific for mouse immunoglobulin IgG₁, IgG_(2a), IgG_(2b), IgG₃, and IgM. Antibodies against hTRPx3 were IgG subclass; predominantly IgG₁ and IgG_(2a) (IgG_(2a)/IgG₁=0.56) suggesting that Th1 and Th2 responses were induced.

Example 3

[0075] The present example demonstrates that immunization with a tri-hybrid melanoma antigen, hTRPx3 protein, induced a T cell immune response.

[0076] To determine if T cells specific for individual antigens were induced in mice, splenocytes from hTRPx3 immunized mice were incubated with hTRP-1, hTRP-2, and tyrosinase protein respectively. The IFNγ released by the T cells in the supernatant was determined by standard IFNγ kits.

[0077] The splenocytes from hTRPx3 immunized mice were found to release significantly higher amounts of IFNγ as compared to control mice following stimulation by antigens (8, 4, and 4-fold increase in IFNγ release upon hTRP-1, hTRP-2, and h-tyrosinase stimulation, respectively). Splenocytes from saline control mice also released IFNγ when stimulated with individual antigens, suggesting these recombinant proteins can have induced some T cell activation in vitro.

[0078] B16 melanoma cells express all tyrosinase family members and MHC class I molecules. T cells specific for hTRPx3 should also react with syngeneic B16 cells. To confirm this, ELISPOT assays were conducted. Briefly, different concentrations of fresh isolated spleen cells from each vaccinated mice group were added to the well of ELISPOT plate. Cells were incubated either with B16 or EL4 control cells. The IFNγ released were captured by mAb to mouse IFNγ on ELISPOT plate.

[0079] As shown in FIG. 5, splenocytes from hTRPx3 immunized mice release IFNγ upon B16 cell stimulation. Since B16 cell did not express MHC class II molecule, it is highly possible that CD8 T cells were responsible for these IFNγ spots.

Example 4

[0080] The present example demonstrates that immunization with a tri-hybrid melanoma antigen, hTPRx3 protein, was useful in treating tumors in mammals.

[0081] To determine the effects of autoimmunity to hTRPx3 on melanoma in vivo, a syngeneic mouse model was used. Ten days after the last booster, immunized C57BL/6 mice were injected intravenously (i.v.) with B16BL6 melanoma cells, which is a spontaneously occurring melanoma from C57BL/6 mouse.

[0082] Mice immunized with recombinant hTRPx3 were significantly protected from lung metastases of B16BL16 melanoma (FIG. 6A). In comparison with control mice, hTRPx3 protein immunized mice had a significant less lung metastases, with 80% (p=0.001) fewer lung nodules.

[0083] To determine the mechanisms of antitumor activity, tumor protection was evaluated following hTRPx3 immunization in MHC class I, class II, and FcR knock-out mice (FIG. 6B). Mice with deficiency in MHC class I have lost tumor protection. Deficiency in MHC class II molecules has no effect on antitumor activity in mice, suggesting that CD8 cells can play a key role in hTRPx3 mediated antitumor activity.

[0084] hTRPx3 immunization was further tested using different adjuvants. Both BCG and GM-CSF have been reported as Th1 driven adjuvants (see, e.g., Disis et al., Blood, 88(1):202-10 (July 1996); Kumar et al., Immunology, 97(3):515-21 (July 1999)). Mice vaccinated with hTRPx3/BCG were protected from melanoma challenge similar to mice immunized with hTRPx3/CFA. Mice vaccinated with hTRPx3/GM-CSF were not protected from tumor challenge.

1 16 1 30 DNA Artificial Sequence Amplification Primer 1 gccgaatcca tgagtgctcc taaactcctc 30 2 63 DNA Artificial Sequence Amplification primer 2 cgtcatgcag actcggggga actgccctcc gccaccctca ggtacactaa actcccgact 60 tgg 63 3 63 DNA Artificial Sequence Amplification primer 3 ccaagtcggg agtttagtgt acctgagggt ggcggagggc agttcccccg agtctgcatg 60 acg 63 4 60 DNA Artificial Sequence Amplification primer 4 ggagacacag gctctaggga aatgtccacc cccgccagtt gtgggccaac ctggagtttc 60 5 60 DNA Artificial Sequence Amplification primer 5 gaaactccag gttggcccac aactggcggg ggtggacatt tccctagagc ctgtgtctcc 60 6 36 DNA Artificial Sequence Amplification primer 6 gcggcgctcg agctatgacc agatccgact cgcttg 36 7 4176 DNA Artificial Sequence Tri-hybrid antigen encoding sequence 7 atg agt gct cct aaa ctc ctc tct ctg ggc tgt atc ttc ttc ccc ttg 48 Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 5 10 15 cta ctt ttt cag cag gcc cgg gct caa ttc cca aga cag tgt gcc act 96 Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 20 25 30 gtt gag gct ttg aga agt ggt atg tgt tgc cca gac ctg tcc cct gtg 144 Val Glu Ala Leu Arg Ser Gly Met Cys Cys Pro Asp Leu Ser Pro Val 35 40 45 tct ggg cct ggg aca gac cgc tgt ggc tca tca tca ggg agg ggc aga 192 Ser Gly Pro Gly Thr Asp Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 50 55 60 tgt gag gca gtg act gca gac tcc cgg ccc cac agc cct cag tat ccc 240 Cys Glu Ala Val Thr Ala Asp Ser Arg Pro His Ser Pro Gln Tyr Pro 65 70 75 80 cat gat ggc aga gat gat cgg gag gtc tgg ccc ttg cgc ttc ttc aat 288 His Asp Gly Arg Asp Asp Arg Glu Val Trp Pro Leu Arg Phe Phe Asn 85 90 95 agg aca tgt cac tgc aac ggc aat ttc tca gga cac aac tgt ggg acg 336 Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 100 105 110 tgc cgt cct ggc tgg aga gga gct gcc tgt gac cag agg gtt ctc ata 384 Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Asp Gln Arg Val Leu Ile 115 120 125 gtc agg aga aat ctt ctg gac tta agt aaa gaa gaa aag aac cac ttt 432 Val Arg Arg Asn Leu Leu Asp Leu Ser Lys Glu Glu Lys Asn His Phe 130 135 140 gtc cgg gcc ctg gat atg gca aag cgc aca act cac cct tta ttt gtc 480 Val Arg Ala Leu Asp Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 145 150 155 160 att gcc acc agg aga tca gaa gaa ata ctg ggg cca gat ggc aac acg 528 Ile Ala Thr Arg Arg Ser Glu Glu Ile Leu Gly Pro Asp Gly Asn Thr 165 170 175 cca caa ttt gag aac att tcc att tat aac tac ttt gtt tgg aca cac 576 Pro Gln Phe Glu Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 180 185 190 tat tac tca gtc aaa aag act ttc ctt ggg gta gga cag gaa agc ttt 624 Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Glu Ser Phe 195 200 205 ggt gaa gtg gat ttc tct cat gag gga cca gct ttt ctc aca tgg cac 672 Gly Glu Val Asp Phe Ser His Glu Gly Pro Ala Phe Leu Thr Trp His 210 215 220 agg tac cac ctc ctg cgt ctg gag aaa gac atg cag gaa atg ttg caa 720 Arg Tyr His Leu Leu Arg Leu Glu Lys Asp Met Gln Glu Met Leu Gln 225 230 235 240 gag cct tct ttc tcc ctt cct tac tgg aat ttt gca acg ggg aaa aat 768 Glu Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 245 250 255 gtc tgt gat atc tgc acg gat gac ttg atg gga tcc aga agc aac ttt 816 Val Cys Asp Ile Cys Thr Asp Asp Leu Met Gly Ser Arg Ser Asn Phe 260 265 270 gat tcc act cta ata agc cca aac tct gtc ttt tct caa tgg cga gtg 864 Asp Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 275 280 285 gtc tgt gac tcc ttg gaa gat tat gat acc ctg gga aca ctt tgt aac 912 Val Cys Asp Ser Leu Glu Asp Tyr Asp Thr Leu Gly Thr Leu Cys Asn 290 295 300 agc acc gag gat ggg cca att agg aga aat cca gct gga aat gtg gcc 960 Ser Thr Glu Asp Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 305 310 315 320 aga cca atg gtg caa cgt ctt cct gaa cca cag gat gtc gct cag tgc 1008 Arg Pro Met Val Gln Arg Leu Pro Glu Pro Gln Asp Val Ala Gln Cys 325 330 335 ttg gaa gtt ggt tta ttt gac acg cct cct ttt tat tcc aac tct aca 1056 Leu Glu Val Gly Leu Phe Asp Thr Pro Pro Phe Tyr Ser Asn Ser Thr 340 345 350 aac agt ttc cga aac aca gtg gaa ggt tac agt gac ccc acg gga aag 1104 Asn Ser Phe Arg Asn Thr Val Glu His Asn Leu Ala His Leu Phe Leu 355 360 365 tat gac cct gct gtt cga agt ctt cac aat ttg gct cat cta ttc ctg 1152 Asn Gly Thr Gly Gly Gln Thr His Gly Tyr Ser Asp Pro Thr Gly Lys 370 375 380 aat gga aca ggg gga caa acc cat ttg tct cca aat gat cct att ttt 1200 Tyr Asp Pro Ala Val Arg Ser Leu Leu Ser Pro Asn Asp Pro Ile Phe 385 390 395 400 gtc ctc ctg cac acc ttc aca gat gca gtc ttt gat gaa tgg ctg agg 1248 Val Leu Leu His Thr Phe Thr Asp Ala Val Phe Asp Glu Trp Leu Arg 405 410 415 aga tac aat gct gat ata tcc aca ttt cca ttg gaa aat gcc cct att 1296 Arg Tyr Asn Ala Asp Ile Ser Thr Phe Pro Leu Glu Asn Ala Pro Ile 420 425 430 gga cat aat aga caa tac aac atg gtg cca ttc tgg ccc cca gtc acc 1344 Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 435 440 445 aac aca gaa atg ttt gtt act gct cca gac aac ctg gga tac act tat 1392 Asn Thr Glu Met Phe Val Thr Ala Pro Asp Asn Leu Gly Tyr Thr Tyr 450 455 460 gaa att caa tgg cca agt cgg gag ttt agt gta cct gag ggt ggc gga 1440 Glu Ile Gln Trp Pro Ser Arg Glu Phe Ser Val Pro Glu Gly Gly Gly 465 470 475 480 ggg cag ttc ccc cga gtc tgc atg acg gtg gac agc cta gtg aac aag 1488 Gly Gln Phe Pro Arg Val Cys Met Thr Val Asp Ser Leu Val Asn Lys 485 490 495 gag tgc tgc cca cgc ctg ggt gca gag tcg gcc aat gtc tgt ggc tct 1536 Glu Cys Cys Pro Arg Leu Gly Ala Glu Ser Ala Asn Val Cys Gly Ser 500 505 510 cag caa ggc cgg ggg cag tgc aca gag gtg cga gcc gac aca agg ccc 1584 Gln Gln Gly Arg Gly Gln Cys Thr Glu Val Arg Ala Asp Thr Arg Pro 515 520 525 tgg agt ggt ccc tac atc cta cga aac cag gat gac cgt gag ctg tgg 1632 Trp Ser Gly Pro Tyr Ile Leu Arg Cys Lys Cys Thr Gly Asn Phe Ala 530 535 540 cca aga aaa ttc ttc cac cgg acc tgc aag tgc aca gga aac ttt gcc 1680 Gly Tyr Asn Cys Gly Asp Cys Lys Asn Gln Asp Asp Arg Glu Leu Trp 545 550 555 560 ggc tat aat tgt gga gac tgc aag ttt ggc tgg acc ggt ccc aac tgc 1728 Pro Arg Lys Phe Phe His Arg Thr Phe Gly Trp Thr Gly Pro Asn Cys 565 570 575 gag cgg aag aaa cca cca gtg att cgg cag aac atc cat tcc ttg agt 1776 Glu Arg Lys Lys Pro Pro Val Ile Arg Gln Asn Ile His Ser Leu Ser 580 585 590 cct cag gaa aga gag cag ttc ttg ggc gcc tta gat ctc gcg aag aag 1824 Pro Gln Glu Arg Glu Gln Phe Leu Gly Ala Leu Asp Leu Ala Lys Lys 595 600 605 aga gta cac ccc gac tac gtg atc acc aca caa cac tgg ctg ggc ctg 1872 Arg Val His Pro Asp Tyr Val Ile Thr Thr Gln His Trp Leu Gly Leu 610 615 620 ctt ggg ccc aat gga acc cag ccg cag ttt gcc aac tgc agt gtt tat 1920 Leu Gly Pro Asn Gly Thr Gln Pro Gln Phe Ala Asn Cys Ser Val Tyr 625 630 635 640 gat ttt ttt gtg tgg ctc cat tat tat tct gtt aga gat aca tta tta 1968 Asp Phe Phe Val Trp Leu His Tyr Tyr Ser Val Arg Asp Thr Leu Leu 645 650 655 gga cca gga cgc ccc tac agg gcc ata gat ttc tca cat caa gga cct 2016 Gly Pro Gly Arg Pro Tyr Arg Ala Ile Asp Phe Ser His Gln Gly Pro 660 665 670 gca ttt gtt acc tgg cac cgg tac cat ttg ttg tgt ctg gaa aga gat 2064 Ala Phe Val Thr Trp His Arg Tyr His Leu Leu Cys Leu Glu Arg Asp 675 680 685 ctc cag cga ctc att ggc aat gag tct ttt gct ttg ccc tac tgg aac 2112 Leu Gln Arg Leu Ile Gly Asn Glu Ser Phe Ala Leu Pro Tyr Trp Asn 690 695 700 ttt gcc act ggg agg aac gag tgt gat gtg tgt aca gac cag ctg ttt 2160 Phe Ala Thr Gly Arg Asn Glu Cys Asp Val Cys Thr Asp Gln Leu Phe 705 710 715 720 ggg gca gcg aga cca gac gat ccg act ctg att agt cgg aac tca aga 2208 Gly Ala Ala Arg Pro Asp Asp Pro Thr Leu Ile Ser Arg Asn Ser Arg 725 730 735 ttc tcc agc tgg gaa act gtc tgt gat agc ttg gat gac tac aac cac 2256 Phe Ser Ser Trp Glu Thr Val Cys Asp Ser Leu Asp Asp Tyr Asn His 740 745 750 ctg gtc acc ttg tgc aat gga acc tat gaa ggt ttg ctg aga aga aat 2304 Leu Val Thr Leu Cys Asn Gly Thr Tyr Glu Gly Leu Leu Arg Arg Asn 755 760 765 caa atg gga aga aac agc atg aaa ttg cca acc tta aaa gac ata cga 2352 Gln Met Gly Arg Asn Ser Met Lys Leu Pro Thr Leu Lys Asp Ile Arg 770 775 780 gat tgc ctg tct ctc cag aag ttt gac aat cct ccc ttc ttc cag aac 2400 Asp Cys Leu Ser Leu Gln Lys Phe Asp Asn Pro Pro Phe Phe Gln Asn 785 790 795 800 tct acc ttc agt ttc agg aat gct ttg gaa ggg ttt gat aaa gca gat 2448 Ser Thr Phe Ser Phe Arg Asn Ala Leu Glu Gly Phe Asp Lys Ala Asp 805 810 815 ggg act ctg gat tct caa gtg atg agc ctt cat aat ttg gtt cat tcc 2496 Gly Thr Leu Asp Ser Gln Val Met Ser Leu His Asn Leu Val His Ser 820 825 830 ttc ctg aac ggg aca aac gct ttg cca cat tca gcc gcc aat gat ccc 2544 Phe Leu Asn Gly Thr Asn Ala Leu Pro His Ser Ala Ala Asn Asp Pro 835 840 845 att ttt gtg gtt ctt cat tcc ttt act gat gcc atc ttt gat gag tgg 2592 Ile Phe Val Val Leu His Ser Phe Thr Asp Ala Ile Phe Asp Glu Trp 850 855 860 atg aaa aga ttt aat cct cct gca gat gcc tgg cct cag gag ctg gcc 2640 Met Lys Arg Phe Asn Pro Pro Ala Asp Ala Trp Pro Gln Glu Leu Ala 865 870 875 880 cct att ggt cac aat cgg atg tac aac atg gtt cct ttc ttc cct cca 2688 Pro Ile Gly His Asn Arg Met Tyr Asn Met Val Pro Phe Phe Pro Pro 885 890 895 gtg act aat gaa gaa ctc ttt tta acc tca gac caa ctt ggc tac agc 2736 Val Thr Asn Glu Glu Leu Phe Leu Thr Ser Asp Gln Leu Gly Tyr Ser 900 905 910 tat gcc atc gat ctg cca gtt tca gtt gaa gaa act cca ggt tgg ccc 2784 Tyr Ala Ile Asp Leu Pro Val Ser Val Glu Glu Thr Pro Gly Trp Pro 915 920 925 aca act ggc ggg ggt gga cat ttc cct aga gcc tgt gtc tcc tct aag 2832 Thr Thr Gly Gly Gly Gly His Phe Pro Arg Ala Cys Val Ser Ser Lys 930 935 940 aac ctg atg gag aag gaa tgc tgt cca ccg tgg agc ggg gac agg agt 2880 Asn Leu Met Glu Lys Glu Cys Cys Pro Pro Trp Ser Gly Asp Arg Ser 945 950 955 960 ccc tgt ggc cag ctt tca ggc aga ggt tcc tgt cag aat atc ctt ctg 2928 Pro Cys Gly Gln Leu Ser Gly Arg Gly Ser Cys Gln Asn Ile Leu Leu 965 970 975 tcc aat gca cca ctt ggg cct caa ttt ccc ttc aca ggg gtg gat gac 2976 Ser Asn Ala Pro Leu Gly Pro Gln Phe Pro Phe Thr Gly Val Asp Asp 980 985 990 cgg gag tcg tgg cct tcc gtc ttt tat aat agg acc tgc cag tgc tct 3024 Arg Glu Ser Trp Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys Ser 995 1000 1005 ggc aac ttc atg gga ttc aac tgt gga aac tgc aag ttt ggc ttt tgg 3072 Gly Asn Phe Met Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Trp 1010 1015 1020 gga cca aac tgc aca gag aga cga ctc ttg gtg aga aga aac atc ttc 3120 Gly Pro Asn Cys Thr Glu Arg Arg Leu Leu Val Arg Arg Asn Ile Phe 1025 1030 1035 1040 gat ttg agt gcc cca gag aag gac aaa ttt ttt gcc tac ctc act tta 3168 Asp Leu Ser Ala Pro Glu Lys Asp Lys Phe Phe Ala Tyr Leu Thr Leu 1045 1050 1055 gca aag cat acc atc agc tca gac tat gtc atc ccc ata ggg acc tat 3216 Ala Lys His Thr Ile Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr 1060 1065 1070 ggc caa atg aaa aat gga tca aca ccc atg ttt aac gac atc aat att 3264 Gly Gln Met Lys Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Ile 1075 1080 1085 tat gac ctc ttt gtc tgg atg cat tat tat gtg tca atg gat gca ctg 3312 Tyr Asp Leu Phe Val Trp Met His Tyr Tyr Val Ser Met Asp Ala Leu 1090 1095 1100 ctt ggg gga tct gaa atc tgg aga gac att gat ttt gcc cat gaa gca 3360 Leu Gly Gly Ser Glu Ile Trp Arg Asp Ile Asp Phe Ala His Glu Ala 1105 1110 1115 1120 cca gct ttt ctg cct tgg cat aga ctc ttc ttg ttg cgg tgg gaa caa 3408 Pro Ala Phe Leu Pro Trp His Arg Leu Phe Leu Leu Arg Trp Glu Gln 1125 1130 1135 gaa atc cag aag ctg aca gga gat gaa aac ttc act att cca tat tgg 3456 Glu Ile Gln Lys Leu Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr Trp 1140 1145 1150 gac tgg cgg gat gca gaa aag tgt gac att tgc aca gat gag tac atg 3504 Asp Trp Arg Asp Ala Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr Met 1155 1160 1165 gga ggt cag cac ccc aca aat cct aac tta ctc agc cca gca tca ttc 3552 Gly Gly Gln His Pro Thr Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe 1170 1175 1180 ttc tcc tct tgg cag att gtc tgt agc cga ttg gag gag tac aac agc 3600 Phe Ser Ser Trp Gln Ile Val Cys Ser Arg Leu Glu Glu Tyr Asn Ser 1185 1190 1195 1200 cat cag tct tta tgc aat gga acg ccc gag gga cct tta cgg cgt aat 3648 His Gln Ser Leu Cys Asn Gly Thr Pro Glu Gly Pro Leu Arg Arg Asn 1205 1210 1215 cct gga aac cat gac aaa tcc aga acc cca agg ctc ccc tct tca gct 3696 Pro Gly Asn His Asp Lys Ser Arg Thr Pro Arg Leu Pro Ser Ser Ala 1220 1225 1230 gat gta gaa ttt tgc ctg agt ttg acc caa tat gaa tct ggt tcc atg 3744 Asp Val Glu Phe Cys Leu Ser Leu Thr Gln Tyr Glu Ser Gly Ser Met 1235 1240 1245 gat aaa gct gcc aat ttc agc ttt aga aat aca ctg gaa gga ttt gct 3792 Asp Lys Ala Ala Asn Phe Ser Phe Arg Asn Thr Leu Glu Gly Phe Ala 1250 1255 1260 agt cca ctt act ggg ata gcg gat gcc tct caa agc agc atg cac aat 3840 Ser Pro Leu Thr Gly Ile Ala Asp Ala Ser Gln Ser Ser Met His Asn 1265 1270 1275 1280 gcc ttg cac atc tat atg aat gga aca atg tcc cag gta cag gga tct 3888 Ala Leu His Ile Tyr Met Asn Gly Thr Met Ser Gln Val Gln Gly Ser 1285 1290 1295 gcc aac gat cct atc ttc ctt ctt cac cat gca ttt gtt gac agt att 3936 Ala Asn Asp Pro Ile Phe Leu Leu His His Ala Phe Val Asp Ser Ile 1300 1305 1310 ttt gag cag tgg ctc cga agg cac cgt cct ctt caa gaa gtt tat cca 3984 Phe Glu Gln Trp Leu Arg Arg His Arg Pro Leu Gln Glu Val Tyr Pro 1315 1320 1325 gaa gcc aat gca ccc att gga cat aac cgg gaa tcc tac atg gtt cct 4032 Glu Ala Asn Ala Pro Ile Gly His Asn Arg Glu Ser Tyr Met Val Pro 1330 1335 1340 ttt ata cca ctg tac aga aat ggt gat ttc ttt att tca tcc aaa gat 4080 Phe Ile Pro Leu Tyr Arg Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp 1345 1350 1355 1360 ctg ggc tat gac tat agc tat cta caa gat tca gac cca gac tct ttt 4128 Leu Gly Tyr Asp Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe 1365 1370 1375 caa gac tac att aag tcc tat ttg gaa caa gcg agt cgg atc tgg tca 4176 Gln Asp Tyr Ile Lys Ser Tyr Leu Glu Gln Ala Ser Arg Ile Trp Ser 1380 1385 1390 8 1392 PRT Artificial Sequence Tri-hybrid antigen 8 Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 5 10 15 Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 20 25 30 Val Glu Ala Leu Arg Ser Gly Met Cys Cys Pro Asp Leu Ser Pro Val 35 40 45 Ser Gly Pro Gly Thr Asp Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 50 55 60 Cys Glu Ala Val Thr Ala Asp Ser Arg Pro His Ser Pro Gln Tyr Pro 65 70 75 80 His Asp Gly Arg Asp Asp Arg Glu Val Trp Pro Leu Arg Phe Phe Asn 85 90 95 Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 100 105 110 Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Asp Gln Arg Val Leu Ile 115 120 125 Val Arg Arg Asn Leu Leu Asp Leu Ser Lys Glu Glu Lys Asn His Phe 130 135 140 Val Arg Ala Leu Asp Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 145 150 155 160 Ile Ala Thr Arg Arg Ser Glu Glu Ile Leu Gly Pro Asp Gly Asn Thr 165 170 175 Pro Gln Phe Glu Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 180 185 190 Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Glu Ser Phe 195 200 205 Gly Glu Val Asp Phe Ser His Glu Gly Pro Ala Phe Leu Thr Trp His 210 215 220 Arg Tyr His Leu Leu Arg Leu Glu Lys Asp Met Gln Glu Met Leu Gln 225 230 235 240 Glu Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 245 250 255 Val Cys Asp Ile Cys Thr Asp Asp Leu Met Gly Ser Arg Ser Asn Phe 260 265 270 Asp Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 275 280 285 Val Cys Asp Ser Leu Glu Asp Tyr Asp Thr Leu Gly Thr Leu Cys Asn 290 295 300 Ser Thr Glu Asp Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 305 310 315 320 Arg Pro Met Val Gln Arg Leu Pro Glu Pro Gln Asp Val Ala Gln Cys 325 330 335 Leu Glu Val Gly Leu Phe Asp Thr Pro Pro Phe Tyr Ser Asn Ser Thr 340 345 350 Asn Ser Phe Arg Asn Thr Val Glu Gly Tyr Ser Asp Pro Thr Gly Lys 355 360 365 Tyr Asp Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu 370 375 380 Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Asp Pro Ile Phe 385 390 395 400 Val Leu Leu His Thr Phe Thr Asp Ala Val Phe Asp Glu Trp Leu Arg 405 410 415 Arg Tyr Asn Ala Asp Ile Ser Thr Phe Pro Leu Glu Asn Ala Pro Ile 420 425 430 Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 435 440 445 Asn Thr Glu Met Phe Val Thr Ala Pro Asp Asn Leu Gly Tyr Thr Tyr 450 455 460 Glu Ile Gln Trp Pro Ser Arg Glu Phe Ser Val Pro Glu Gly Gly Gly 465 470 475 480 Gly Gln Phe Pro Arg Val Cys Met Thr Val Asp Ser Leu Val Asn Lys 485 490 495 Glu Cys Cys Pro Arg Leu Gly Ala Glu Ser Ala Asn Val Cys Gly Ser 500 505 510 Gln Gln Gly Arg Gly Gln Cys Thr Glu Val Arg Ala Asp Thr Arg Pro 515 520 525 Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln Asp Asp Arg Glu Leu Trp 530 535 540 Pro Arg Lys Phe Phe His Arg Thr Cys Lys Cys Thr Gly Asn Phe Ala 545 550 555 560 Gly Tyr Asn Cys Gly Asp Cys Lys Phe Gly Trp Thr Gly Pro Asn Cys 565 570 575 Glu Arg Lys Lys Pro Pro Val Ile Arg Gln Asn Ile His Ser Leu Ser 580 585 590 Pro Gln Glu Arg Glu Gln Phe Leu Gly Ala Leu Asp Leu Ala Lys Lys 595 600 605 Arg Val His Pro Asp Tyr Val Ile Thr Thr Gln His Trp Leu Gly Leu 610 615 620 Leu Gly Pro Asn Gly Thr Gln Pro Gln Phe Ala Asn Cys Ser Val Tyr 625 630 635 640 Asp Phe Phe Val Trp Leu His Tyr Tyr Ser Val Arg Asp Thr Leu Leu 645 650 655 Gly Pro Gly Arg Pro Tyr Arg Ala Ile Asp Phe Ser His Gln Gly Pro 660 665 670 Ala Phe Val Thr Trp His Arg Tyr His Leu Leu Cys Leu Glu Arg Asp 675 680 685 Leu Gln Arg Leu Ile Gly Asn Glu Ser Phe Ala Leu Pro Tyr Trp Asn 690 695 700 Phe Ala Thr Gly Arg Asn Glu Cys Asp Val Cys Thr Asp Gln Leu Phe 705 710 715 720 Gly Ala Ala Arg Pro Asp Asp Pro Thr Leu Ile Ser Arg Asn Ser Arg 725 730 735 Phe Ser Ser Trp Glu Thr Val Cys Asp Ser Leu Asp Asp Tyr Asn His 740 745 750 Leu Val Thr Leu Cys Asn Gly Thr Tyr Glu Gly Leu Leu Arg Arg Asn 755 760 765 Gln Met Gly Arg Asn Ser Met Lys Leu Pro Thr Leu Lys Asp Ile Arg 770 775 780 Asp Cys Leu Ser Leu Gln Lys Phe Asp Asn Pro Pro Phe Phe Gln Asn 785 790 795 800 Ser Thr Phe Ser Phe Arg Asn Ala Leu Glu Gly Phe Asp Lys Ala Asp 805 810 815 Gly Thr Leu Asp Ser Gln Val Met Ser Leu His Asn Leu Val His Ser 820 825 830 Phe Leu Asn Gly Thr Asn Ala Leu Pro His Ser Ala Ala Asn Asp Pro 835 840 845 Ile Phe Val Val Leu His Ser Phe Thr Asp Ala Ile Phe Asp Glu Trp 850 855 860 Met Lys Arg Phe Asn Pro Pro Ala Asp Ala Trp Pro Gln Glu Leu Ala 865 870 875 880 Pro Ile Gly His Asn Arg Met Tyr Asn Met Val Pro Phe Phe Pro Pro 885 890 895 Val Thr Asn Glu Glu Leu Phe Leu Thr Ser Asp Gln Leu Gly Tyr Ser 900 905 910 Tyr Ala Ile Asp Leu Pro Val Ser Val Glu Glu Thr Pro Gly Trp Pro 915 920 925 Thr Thr Gly Gly Gly Gly His Phe Pro Arg Ala Cys Val Ser Ser Lys 930 935 940 Asn Leu Met Glu Lys Glu Cys Cys Pro Pro Trp Ser Gly Asp Arg Ser 945 950 955 960 Pro Cys Gly Gln Leu Ser Gly Arg Gly Ser Cys Gln Asn Ile Leu Leu 965 970 975 Ser Asn Ala Pro Leu Gly Pro Gln Phe Pro Phe Thr Gly Val Asp Asp 980 985 990 Arg Glu Ser Trp Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys Ser 995 1000 1005 Gly Asn Phe Met Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Trp 1010 1015 1020 Gly Pro Asn Cys Thr Glu Arg Arg Leu Leu Val Arg Arg Asn Ile Phe 1025 1030 1035 1040 Asp Leu Ser Ala Pro Glu Lys Asp Lys Phe Phe Ala Tyr Leu Thr Leu 1045 1050 1055 Ala Lys His Thr Ile Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr 1060 1065 1070 Gly Gln Met Lys Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Ile 1075 1080 1085 Tyr Asp Leu Phe Val Trp Met His Tyr Tyr Val Ser Met Asp Ala Leu 1090 1095 1100 Leu Gly Gly Ser Glu Ile Trp Arg Asp Ile Asp Phe Ala His Glu Ala 1105 1110 1115 1120 Pro Ala Phe Leu Pro Trp His Arg Leu Phe Leu Leu Arg Trp Glu Gln 1125 1130 1135 Glu Ile Gln Lys Leu Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr Trp 1140 1145 1150 Asp Trp Arg Asp Ala Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr Met 1155 1160 1165 Gly Gly Gln His Pro Thr Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe 1170 1175 1180 Phe Ser Ser Trp Gln Ile Val Cys Ser Arg Leu Glu Glu Tyr Asn Ser 1185 1190 1195 1200 His Gln Ser Leu Cys Asn Gly Thr Pro Glu Gly Pro Leu Arg Arg Asn 1205 1210 1215 Pro Gly Asn His Asp Lys Ser Arg Thr Pro Arg Leu Pro Ser Ser Ala 1220 1225 1230 Asp Val Glu Phe Cys Leu Ser Leu Thr Gln Tyr Glu Ser Gly Ser Met 1235 1240 1245 Asp Lys Ala Ala Asn Phe Ser Phe Arg Asn Thr Leu Glu Gly Phe Ala 1 250 1255 1260 Ser Pro Leu Thr Gly Ile Ala Asp Ala Ser Gln Ser Ser Met His Asn 1265 1270 1275 1280 Ala Leu His Ile Tyr Met Asn Gly Thr Met Ser Gln Val Gln Gly Ser 1285 1290 1295 Ala Asn Asp Pro Ile Phe Leu Leu His His Ala Phe Val Asp Ser Ile 1300 1305 1310 Phe Glu Gln Trp Leu Arg Arg His Arg Pro Leu Gln Glu Val Tyr Pro 1315 1320 1325 Glu Ala Asn Ala Pro Ile Gly His Asn Arg Glu Ser Tyr Met Val Pro 1330 1335 1340 Phe Ile Pro Leu Tyr Arg Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp 1345 1350 1355 1360 Leu Gly Tyr Asp Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe 1365 1370 1375 Gln Asp Tyr Ile Lys Ser Tyr Leu Glu Gln Ala Ser Arg Ile Trp Ser 1380 1385 1390 9 4104 DNA Artificial Sequence Tri-hybrid antigen coding sequence 9 caa ttc cca aga cag tgt gcc act gtt gag gct ttg aga agt ggt atg 48 Gln Phe Pro Arg Gln Cys Ala Thr Val Glu Ala Leu Arg Ser Gly Met 5 10 15 tgt tgc cca gac ctg tcc cct gtg tct ggg cct ggg aca gac cgc tgt 96 Cys Cys Pro Asp Leu Ser Pro Val Ser Gly Pro Gly Thr Asp Arg Cys 20 25 30 ggc tca tca tca ggg agg ggc aga tgt gag gca gtg act gca gac tcc 144 Gly Ser Ser Ser Gly Arg Gly Arg Cys Glu Ala Val Thr Ala Asp Ser 35 40 45 cgg ccc cac agc cct cag tat ccc cat gat ggc aga gat gat cgg gag 192 Arg Pro His Ser Pro Gln Tyr Pro His Asp Gly Arg Asp Asp Arg Glu 50 55 60 gtc tgg ccc ttg cgc ttc ttc aat agg aca tgt cac tgc aac ggc aat 240 Val Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys His Cys Asn Gly Asn 65 70 75 80 ttc tca gga cac aac tgt ggg acg tgc cgt cct ggc tgg aga gga gct 288 Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala 85 90 95 gcc tgt gac cag agg gtt ctc ata gtc agg aga aat ctt ctg gac tta 336 Ala Cys Asp Gln Arg Val Leu Ile Val Arg Arg Asn Leu Leu Asp Leu 100 105 110 agt aaa gaa gaa aag aac cac ttt gtc cgg gcc ctg gat atg gca aag 384 Ser Lys Glu Glu Lys Asn His Phe Val Arg Ala Leu Asp Met Ala Lys 115 120 125 cgc aca act cac cct tta ttt gtc att gcc acc agg aga tca gaa gaa 432 Arg Thr Thr His Pro Leu Phe Val Ile Ala Thr Arg Arg Ser Glu Glu 130 135 140 ata ctg ggg cca gat ggc aac acg cca caa ttt gag aac att tcc att 480 Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Ile 145 150 155 160 tat aac tac ttt gtt tgg aca cac tat tac tca gtc aaa aag act ttc 528 Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe 165 170 175 ctt ggg gta gga cag gaa agc ttt ggt gaa gtg gat ttc tct cat gag 576 Leu Gly Val Gly Gln Glu Ser Phe Gly Glu Val Asp Phe Ser His Glu 180 185 190 gga cca gct ttt ctc aca tgg cac agg tac cac ctc ctg cgt ctg gag 624 Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Arg Leu Glu 195 200 205 aaa gac atg cag gaa atg ttg caa gag cct tct ttc tcc ctt cct tac 672 Lys Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr 210 215 220 tgg aat ttt gca acg ggg aaa aat gtc tgt gat atc tgc acg gat gac 720 Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Ile Cys Thr Asp Asp 225 230 235 240 ttg atg gga tcc aga agc aac ttt gat tcc act cta ata agc cca aac 768 Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn 245 250 255 tct gtc ttt tct caa tgg cga gtg gtc tgt gac tcc ttg gaa gat tat 816 Ser Val Phe Ser Gln Trp Arg Val Val Cys Asp Ser Leu Glu Asp Tyr 260 265 270 gat acc ctg gga aca ctt tgt aac agc acc gag gat ggg cca att agg 864 Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Asp Gly Pro Ile Arg 275 280 285 aga aat cca gct gga aat gtg gcc aga cca atg gtg caa cgt ctt cct 912 Arg Asn Pro Ala Gly Asn Val Ala Arg Pro Met Val Gln Arg Leu Pro 290 295 300 gaa cca cag gat gtc gct cag tgc ttg gaa gtt ggt tta ttt gac acg 960 Glu Pro Gln Asp Val Ala Gln Cys Leu Glu Val Gly Leu Phe Asp Thr 305 310 315 320 cct cct ttt tat tcc aac tct aca aac agt ttc cga aac aca gtg gaa 1008 Pro Pro Phe Tyr Ser Asn Ser Thr Asn Ser Phe Arg Asn Thr Val Glu 325 330 335 ggt tac agt gac ccc acg gga aag tat gac cct gct gtt cga agt ctt 1056 His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His 340 345 350 cac aat ttg gct cat cta ttc ctg aat gga aca ggg gga caa acc cat 1104 Gly Tyr Ser Asp Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu 355 360 365 ttg tct cca aat gat cct att ttt gtc ctc ctg cac acc ttc aca gat 1152 Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp 370 375 380 gca gtc ttt gat gaa tgg ctg agg aga tac aat gct gat ata tcc aca 1200 Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr 385 390 395 400 ttt cca ttg gaa aat gcc cct att gga cat aat aga caa tac aac atg 1248 Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met 405 410 415 gtg cca ttc tgg ccc cca gtc acc aac aca gaa atg ttt gtt act gct 1296 Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala 420 425 430 cca gac aac ctg gga tac act tat gaa att caa tgg cca agt cgg gag 1344 Pro Asp Asn Leu Gly Tyr Thr Tyr Glu Ile Gln Trp Pro Ser Arg Glu 435 440 445 ttt agt gta cct gag ggt ggc gga ggg cag ttc ccc cga gtc tgc atg 1392 Phe Ser Val Pro Glu Gly Gly Gly Gly Gln Phe Pro Arg Val Cys Met 450 455 460 acg gtg gac agc cta gtg aac aag gag tgc tgc cca cgc ctg ggt gca 1440 Thr Val Asp Ser Leu Val Asn Lys Glu Cys Cys Pro Arg Leu Gly Ala 465 470 475 480 gag tcg gcc aat gtc tgt ggc tct cag caa ggc cgg ggg cag tgc aca 1488 Glu Ser Ala Asn Val Cys Gly Ser Gln Gln Gly Arg Gly Gln Cys Thr 485 490 495 gag gtg cga gcc gac aca agg ccc tgg agt ggt ccc tac atc cta cga 1536 Glu Val Arg Ala Asp Thr Arg Pro Trp Ser Gly Pro Tyr Ile Leu Arg 500 505 510 aac cag gat gac cgt gag ctg tgg cca aga aaa ttc ttc cac cgg acc 1584 Cys Lys Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Asp Cys Lys 515 520 525 tgc aag tgc aca gga aac ttt gcc ggc tat aat tgt gga gac tgc aag 1632 Asn Gln Asp Asp Arg Glu Leu Trp Pro Arg Lys Phe Phe His Arg Thr 530 535 540 ttt ggc tgg acc ggt ccc aac tgc gag cgg aag aaa cca cca gtg att 1680 Phe Gly Trp Thr Gly Pro Asn Cys Glu Arg Lys Lys Pro Pro Val Ile 545 550 555 560 cgg cag aac atc cat tcc ttg agt cct cag gaa aga gag cag ttc ttg 1728 Arg Gln Asn Ile His Ser Leu Ser Pro Gln Glu Arg Glu Gln Phe Leu 565 570 575 ggc gcc tta gat ctc gcg aag aag aga gta cac ccc gac tac gtg atc 1776 Gly Ala Leu Asp Leu Ala Lys Lys Arg Val His Pro Asp Tyr Val Ile 580 585 590 acc aca caa cac tgg ctg ggc ctg ctt ggg ccc aat gga acc cag ccg 1824 Thr Thr Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro 595 600 605 cag ttt gcc aac tgc agt gtt tat gat ttt ttt gtg tgg ctc cat tat 1872 Gln Phe Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr 610 615 620 tat tct gtt aga gat aca tta tta gga cca gga cgc ccc tac agg gcc 1920 Tyr Ser Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Arg Ala 625 630 635 640 ata gat ttc tca cat caa gga cct gca ttt gtt acc tgg cac cgg tac 1968 Ile Asp Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His Arg Tyr 645 650 655 cat ttg ttg tgt ctg gaa aga gat ctc cag cga ctc att ggc aat gag 2016 His Leu Leu Cys Leu Glu Arg Asp Leu Gln Arg Leu Ile Gly Asn Glu 660 665 670 tct ttt gct ttg ccc tac tgg aac ttt gcc act ggg agg aac gag tgt 2064 Ser Phe Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly Arg Asn Glu Cys 675 680 685 gat gtg tgt aca gac cag ctg ttt ggg gca gcg aga cca gac gat ccg 2112 Asp Val Cys Thr Asp Gln Leu Phe Gly Ala Ala Arg Pro Asp Asp Pro 690 695 700 act ctg att agt cgg aac tca aga ttc tcc agc tgg gaa act gtc tgt 2160 Thr Leu Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp Glu Thr Val Cys 705 710 715 720 gat agc ttg gat gac tac aac cac ctg gtc acc ttg tgc aat gga acc 2208 Asp Ser Leu Asp Asp Tyr Asn His Leu Val Thr Leu Cys Asn Gly Thr 725 730 735 tat gaa ggt ttg ctg aga aga aat caa atg gga aga aac agc atg aaa 2256 Tyr Glu Gly Leu Leu Arg Arg Asn Gln Met Gly Arg Asn Ser Met Lys 740 745 750 ttg cca acc tta aaa gac ata cga gat tgc ctg tct ctc cag aag ttt 2304 Leu Pro Thr Leu Lys Asp Ile Arg Asp Cys Leu Ser Leu Gln Lys Phe 755 760 765 gac aat cct ccc ttc ttc cag aac tct acc ttc agt ttc agg aat gct 2352 Asp Asn Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser Phe Arg Asn Ala 770 775 780 ttg gaa ggg ttt gat aaa gca gat ggg act ctg gat tct caa gtg atg 2400 Leu Glu Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met 785 790 795 800 agc ctt cat aat ttg gtt cat tcc ttc ctg aac ggg aca aac gct ttg 2448 Ser Leu His Asn Leu Val His Ser Phe Leu Asn Gly Thr Asn Ala Leu 805 810 815 cca cat tca gcc gcc aat gat ccc att ttt gtg gtt ctt cat tcc ttt 2496 Pro His Ser Ala Ala Asn Asp Pro Ile Phe Val Val Leu His Ser Phe 820 825 830 act gat gcc atc ttt gat gag tgg atg aaa aga ttt aat cct cct gca 2544 Thr Asp Ala Ile Phe Asp Glu Trp Met Lys Arg Phe Asn Pro Pro Ala 835 840 845 gat gcc tgg cct cag gag ctg gcc cct att ggt cac aat cgg atg tac 2592 Asp Ala Trp Pro Gln Glu Leu Ala Pro Ile Gly His Asn Arg Met Tyr 850 855 860 aac atg gtt cct ttc ttc cct cca gtg act aat gaa gaa ctc ttt tta 2640 Asn Met Val Pro Phe Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu 865 870 875 880 acc tca gac caa ctt ggc tac agc tat gcc atc gat ctg cca gtt tca 2688 Thr Ser Asp Gln Leu Gly Tyr Ser Tyr Ala Ile Asp Leu Pro Val Ser 885 890 895 gtt gaa gaa act cca ggt tgg ccc aca act ggc ggg ggt gga cat ttc 2736 Val Glu Glu Thr Pro Gly Trp Pro Thr Thr Gly Gly Gly Gly His Phe 900 905 910 cct aga gcc tgt gtc tcc tct aag aac ctg atg gag aag gaa tgc tgt 2784 Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Glu Lys Glu Cys Cys 915 920 925 cca ccg tgg agc ggg gac agg agt ccc tgt ggc cag ctt tca ggc aga 2832 Pro Pro Trp Ser Gly Asp Arg Ser Pro Cys Gly Gln Leu Ser Gly Arg 930 935 940 ggt tcc tgt cag aat atc ctt ctg tcc aat gca cca ctt ggg cct caa 2880 Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln 945 950 955 960 ttt ccc ttc aca ggg gtg gat gac cgg gag tcg tgg cct tcc gtc ttt 2928 Phe Pro Phe Thr Gly Val Asp Asp Arg Glu Ser Trp Pro Ser Val Phe 965 970 975 tat aat agg acc tgc cag tgc tct ggc aac ttc atg gga ttc aac tgt 2976 Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met Gly Phe Asn Cys 980 985 990 gga aac tgc aag ttt ggc ttt tgg gga cca aac tgc aca gag aga cga 3024 Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys Thr Glu Arg Arg 995 1000 1005 ctc ttg gtg aga aga aac atc ttc gat ttg agt gcc cca gag aag gac 3072 Leu Leu Val Arg Arg Asn Ile Phe Asp Leu Ser Ala Pro Glu Lys Asp 1010 1015 1020 aaa ttt ttt gcc tac ctc act tta gca aag cat acc atc agc tca gac 3120 Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr Ile Ser Ser Asp 1025 1030 1035 1040 tat gtc atc ccc ata ggg acc tat ggc caa atg aaa aat gga tca aca 3168 Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys Asn Gly Ser Thr 1045 1050 1055 ccc atg ttt aac gac atc aat att tat gac ctc ttt gtc tgg atg cat 3216 Pro Met Phe Asn Asp Ile Asn Ile Tyr Asp Leu Phe Val Trp Met His 1060 1065 1070 tat tat gtg tca atg gat gca ctg ctt ggg gga tct gaa atc tgg aga 3264 Tyr Tyr Val Ser Met Asp Ala Leu Leu Gly Gly Ser Glu Ile Trp Arg 1075 1080 1085 gac att gat ttt gcc cat gaa gca cca gct ttt ctg cct tgg cat aga 3312 Asp Ile Asp Phe Ala His Glu Ala Pro Ala Phe Leu Pro Trp His Arg 1090 1095 1100 ctc ttc ttg ttg cgg tgg gaa caa gaa atc cag aag ctg aca gga gat 3360 Leu Phe Leu Leu Arg Trp Glu Gln Glu Ile Gln Lys Leu Thr Gly Asp 1105 1110 1115 1120 gaa aac ttc act att cca tat tgg gac tgg cgg gat gca gaa aag tgt 3408 Glu Asn Phe Thr Ile Pro Tyr Trp Asp Trp Arg Asp Ala Glu Lys Cys 1125 1130 1135 gac att tgc aca gat gag tac atg gga ggt cag cac ccc aca aat cct 3456 Asp Ile Cys Thr Asp Glu Tyr Met Gly Gly Gln His Pro Thr Asn Pro 1140 1145 1150 aac tta ctc agc cca gca tca ttc ttc tcc tct tgg cag att gtc tgt 3504 Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp Gln Ile Val Cys 1155 1160 1165 agc cga ttg gag gag tac aac agc cat cag tct tta tgc aat gga acg 3552 Ser Arg Leu Glu Glu Tyr Asn Ser His Gln Ser Leu Cys Asn Gly Thr 1170 1175 1180 ccc gag gga cct tta cgg cgt aat cct gga aac cat gac aaa tcc aga 3600 Pro Glu Gly Pro Leu Arg Arg Asn Pro Gly Asn His Asp Lys Ser Arg 1185 1190 1195 1200 acc cca agg ctc ccc tct tca gct gat gta gaa ttt tgc ctg agt ttg 3648 Thr Pro Arg Leu Pro Ser Ser Ala Asp Val Glu Phe Cys Leu Ser Leu 1205 1210 1215 acc caa tat gaa tct ggt tcc atg gat aaa gct gcc aat ttc agc ttt 3696 Thr Gln Tyr Glu Ser Gly Ser Met Asp Lys Ala Ala Asn Phe Ser Phe 1220 1225 1230 aga aat aca ctg gaa gga ttt gct agt cca ctt act ggg ata gcg gat 3744 Arg Asn Thr Leu Glu Gly Phe Ala Ser Pro Leu Thr Gly Ile Ala Asp 1235 1240 1245 gcc tct caa agc agc atg cac aat gcc ttg cac atc tat atg aat gga 3792 Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile Tyr Met Asn Gly 1250 1255 1260 aca atg tcc cag gta cag gga tct gcc aac gat cct atc ttc ctt ctt 3840 Thr Met Ser Gln Val Gln Gly Ser Ala Asn Asp Pro Ile Phe Leu Leu 1265 1270 1275 1280 cac cat gca ttt gtt gac agt att ttt gag cag tgg ctc cga agg cac 3888 His His Ala Phe Val Asp Ser Ile Phe Glu Gln Trp Leu Arg Arg His 1285 1290 1295 cgt cct ctt caa gaa gtt tat cca gaa gcc aat gca ccc att gga cat 3936 Arg Pro Leu Gln Glu Val Tyr Pro Glu Ala Asn Ala Pro Ile Gly His 1300 1305 1310 aac cgg gaa tcc tac atg gtt cct ttt ata cca ctg tac aga aat ggt 3984 Asn Arg Glu Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr Arg Asn Gly 1315 1320 1325 gat ttc ttt att tca tcc aaa gat ctg ggc tat gac tat agc tat cta 4032 Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp Tyr Ser Tyr Leu 1330 1335 1340 caa gat tca gac cca gac tct ttt caa gac tac att aag tcc tat ttg 4080 Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr Ile Lys Ser Tyr Leu 1345 1350 1355 1360 gaa caa gcg agt cgg atc tgg tca 4104 Glu Gln Ala Ser Arg Ile Trp Ser 1365 10 1368 PRT Artificial Sequence Tri-hybrid antigen 10 Gln Phe Pro Arg Gln Cys Ala Thr Val Glu Ala Leu Arg Ser Gly Met 5 10 15 Cys Cys Pro Asp Leu Ser Pro Val Ser Gly Pro Gly Thr Asp Arg Cys 20 25 30 Gly Ser Ser Ser Gly Arg Gly Arg Cys Glu Ala Val Thr Ala Asp Ser 35 40 45 Arg Pro His Ser Pro Gln Tyr Pro His Asp Gly Arg Asp Asp Arg Glu 50 55 60 Val Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys His Cys Asn Gly Asn 65 70 75 80 Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala 85 90 95 Ala Cys Asp Gln Arg Val Leu Ile Val Arg Arg Asn Leu Leu Asp Leu 100 105 110 Ser Lys Glu Glu Lys Asn His Phe Val Arg Ala Leu Asp Met Ala Lys 115 120 125 Arg Thr Thr His Pro Leu Phe Val Ile Ala Thr Arg Arg Ser Glu Glu 130 135 140 Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Ile 145 150 155 160 Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe 165 170 175 Leu Gly Val Gly Gln Glu Ser Phe Gly Glu Val Asp Phe Ser His Glu 180 185 190 Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Arg Leu Glu 195 200 205 Lys Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr 210 215 220 Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Ile Cys Thr Asp Asp 225 230 235 240 Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn 245 250 255 Ser Val Phe Ser Gln Trp Arg Val Val Cys Asp Ser Leu Glu Asp Tyr 260 265 270 Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Asp Gly Pro Ile Arg 275 280 285 Arg Asn Pro Ala Gly Asn Val Ala Arg Pro Met Val Gln Arg Leu Pro 290 295 300 Glu Pro Gln Asp Val Ala Gln Cys Leu Glu Val Gly Leu Phe Asp Thr 305 310 315 320 Pro Pro Phe Tyr Ser Asn Ser Thr Asn Ser Phe Arg Asn Thr Val Glu 325 330 335 Gly Tyr Ser Asp Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu 340 345 350 His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His 355 360 365 Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp 370 375 380 Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr 385 390 395 400 Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met 405 410 415 Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala 420 425 430 Pro Asp Asn Leu Gly Tyr Thr Tyr Glu Ile Gln Trp Pro Ser Arg Glu 435 440 445 Phe Ser Val Pro Glu Gly Gly Gly Gly Gln Phe Pro Arg Val Cys Met 450 455 460 Thr Val Asp Ser Leu Val Asn Lys Glu Cys Cys Pro Arg Leu Gly Ala 465 470 475 480 Glu Ser Ala Asn Val Cys Gly Ser Gln Gln Gly Arg Gly Gln Cys Thr 485 490 495 Glu Val Arg Ala Asp Thr Arg Pro Trp Ser Gly Pro Tyr Ile Leu Arg 500 505 510 Asn Gln Asp Asp Arg Glu Leu Trp Pro Arg Lys Phe Phe His Arg Thr 515 520 525 Cys Lys Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Asp Cys Lys 530 535 540 Phe Gly Trp Thr Gly Pro Asn Cys Glu Arg Lys Lys Pro Pro Val Ile 545 550 555 560 Arg Gln Asn Ile His Ser Leu Ser Pro Gln Glu Arg Glu Gln Phe Leu 565 570 575 Gly Ala Leu Asp Leu Ala Lys Lys Arg Val His Pro Asp Tyr Val Ile 580 585 590 Thr Thr Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro 595 600 605 Gln Phe Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr 610 615 620 Tyr Ser Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Arg Ala 625 630 635 640 Ile Asp Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His Arg Tyr 645 650 655 His Leu Leu Cys Leu Glu Arg Asp Leu Gln Arg Leu Ile Gly Asn Glu 660 665 670 Ser Phe Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly Arg Asn Glu Cys 675 680 685 Asp Val Cys Thr Asp Gln Leu Phe Gly Ala Ala Arg Pro Asp Asp Pro 690 695 700 Thr Leu Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp Glu Thr Val Cys 705 710 715 720 Asp Ser Leu Asp Asp Tyr Asn His Leu Val Thr Leu Cys Asn Gly Thr 725 730 735 Tyr Glu Gly Leu Leu Arg Arg Asn Gln Met Gly Arg Asn Ser Met Lys 740 745 750 Leu Pro Thr Leu Lys Asp Ile Arg Asp Cys Leu Ser Leu Gln Lys Phe 755 760 765 Asp Asn Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser Phe Arg Asn Ala 770 775 780 Leu Glu Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met 785 790 795 800 Ser Leu His Asn Leu Val His Ser Phe Leu Asn Gly Thr Asn Ala Leu 805 810 815 Pro His Ser Ala Ala Asn Asp Pro Ile Phe Val Val Leu His Ser Phe 820 825 830 Thr Asp Ala Ile Phe Asp Glu Trp Met Lys Arg Phe Asn Pro Pro Ala 835 840 845 Asp Ala Trp Pro Gln Glu Leu Ala Pro Ile Gly His Asn Arg Met Tyr 850 855 860 Asn Met Val Pro Phe Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu 865 870 875 880 Thr Ser Asp Gln Leu Gly Tyr Ser Tyr Ala Ile Asp Leu Pro Val Ser 885 890 895 Val Glu Glu Thr Pro Gly Trp Pro Thr Thr Gly Gly Gly Gly His Phe 900 905 910 Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Glu Lys Glu Cys Cys 915 920 925 Pro Pro Trp Ser Gly Asp Arg Ser Pro Cys Gly Gln Leu Ser Gly Arg 930 935 940 Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln 945 950 955 960 Phe Pro Phe Thr Gly Val Asp Asp Arg Glu Ser Trp Pro Ser Val Phe 965 970 975 Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met Gly Phe Asn Cys 980 985 990 Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys Thr Glu Arg Arg 995 1000 1005 Leu Leu Val Arg Arg Asn Ile Phe Asp Leu Ser Ala Pro Glu Lys Asp 1010 1015 1020 Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr Ile Ser Ser Asp 1025 1030 1035 1040 Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys Asn Gly Ser Thr 1045 1050 1055 Pro Met Phe Asn Asp Ile Asn Ile Tyr Asp Leu Phe Val Trp Met His 1060 1065 1070 Tyr Tyr Val Ser Met Asp Ala Leu Leu Gly Gly Ser Glu Ile Trp Arg 1075 1080 1085 Asp Ile Asp Phe Ala His Glu Ala Pro Ala Phe Leu Pro Trp His Arg 1090 1095 1100 Leu Phe Leu Leu Arg Trp Glu Gln Glu Ile Gln Lys Leu Thr Gly Asp 1105 1110 1115 1120 Glu Asn Phe Thr Ile Pro Tyr Trp Asp Trp Arg Asp Ala Glu Lys Cys 1125 1130 1135 Asp Ile Cys Thr Asp Glu Tyr Met Gly Gly Gln His Pro Thr Asn Pro 1140 1145 1150 Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp Gln Ile Val Cys 1155 1160 1165 Ser Arg Leu Glu Glu Tyr Asn Ser His Gln Ser Leu Cys Asn Gly Thr 1170 1175 1180 Pro Glu Gly Pro Leu Arg Arg Asn Pro Gly Asn His Asp Lys Ser Arg 1185 1190 1195 1200 Thr Pro Arg Leu Pro Ser Ser Ala Asp Val Glu Phe Cys Leu Ser Leu 1205 1210 1215 Thr Gln Tyr Glu Ser Gly Ser Met Asp Lys Ala Ala Asn Phe Ser Phe 1220 1225 1230 Arg Asn Thr Leu Glu Gly Phe Ala Ser Pro Leu Thr Gly Ile Ala Asp 1235 1240 1245 Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile Tyr Met Asn Gly 1250 1255 1260 Thr Met Ser Gln Val Gln Gly Ser Ala Asn Asp Pro Ile Phe Leu Leu 1265 1270 1275 1280 His His Ala Phe Val Asp Ser Ile Phe Glu Gln Trp Leu Arg Arg His 1285 1290 1295 Arg Pro Leu Gln Glu Val Tyr Pro Glu Ala Asn Ala Pro Ile Gly His 1300 1305 1310 Asn Arg Glu Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr Arg Asn Gly 1315 1320 1325 Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp Tyr Ser Tyr Leu 1330 1335 1340 Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr Ile Lys Ser Tyr Leu 1345 1350 1355 1360 Glu Gln Ala Ser Arg Ile Trp Ser 1365 11 4152 DNA Artificial Sequence Tri-hybrid antigen coding sequence 11 atg agt gct cct aaa ctc ctc tct ctg ggc tgt atc ttc ttc ccc ttg 48 Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 5 10 15 cta ctt ttt cag cag gcc cgg gct caa ttc cca aga cag tgt gcc act 96 Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 20 25 30 gtt gag gct ttg aga agt ggt atg tgt tgc cca gac ctg tcc cct gtg 144 Val Gln Ala Leu Arg Ser Gly Met Cys Cys Pro Glu Leu Ser Pro Val 35 40 45 tct ggg cct ggg aca gac cgc tgt ggc tca tca tca ggg agg ggc aga 192 Ser Gly Pro Gly Thr Glu Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 50 55 60 tgt gag gca gtg act gca gac tcc cgg ccc cac agc cct cag tat ccc 240 Cys Gln Ala Val Thr Ala Glu Ser Arg Pro His Ser Pro Gln Tyr Pro 65 70 75 80 cat gat ggc aga gat gat cgg gag gtc tgg ccc ttg cgc ttc ttc aat 288 His Glu Gly Arg Glu Glu Arg Gln Val Trp Pro Leu Arg Phe Phe Asn 85 90 95 agg aca tgt cac tgc aac ggc aat ttc tca gga cac aac tgt ggg acg 336 Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 100 105 110 tgc cgt cct ggc tgg aga gga gct gcc tgt gac cag agg gtt ctc ata 384 Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Glu Gln Arg Val Leu Ile 115 120 125 gtc agg aga aat ctt ctg gac tta agt aaa gaa gaa aag aac cac ttt 432 Val Arg Arg Asn Leu Leu Glu Leu Ser Lys Gln Gln Lys Asn His Phe 130 135 140 gtc cgg gcc ctg gat atg gca aag cgc aca act cac cct tta ttt gtc 480 Val Arg Ala Leu Glu Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 145 150 155 160 att gcc acc agg aga tca gaa gaa ata ctg ggg cca gat ggc aac acg 528 Ile Ala Thr Arg Arg Ser Gln Gln Ile Leu Gly Pro Glu Gly Asn Thr 165 170 175 cca caa ttt gag aac att tcc att tat aac tac ttt gtt tgg aca cac 576 Pro Gln Phe Gln Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 180 185 190 tat tac tca gtc aaa aag act ttc ctt ggg gta gga cag gaa agc ttt 624 Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Gln Ser Phe 195 200 205 ggt gaa gtg gat ttc tct cat gag gga cca gct ttt ctc aca tgg cac 672 Gly Gln Val Glu Phe Ser His Gln Gly Pro Ala Phe Leu Thr Trp His 210 215 220 agg tac cac ctc ctg cgt ctg gag aaa gac atg cag gaa atg ttg caa 720 Arg Tyr His Leu Leu Arg Leu Gln Lys Glu Met Gln Gln Met Leu Gln 225 230 235 240 gag cct tct ttc tcc ctt cct tac tgg aat ttt gca acg ggg aaa aat 768 Gln Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 245 250 255 gtc tgt gat atc tgc acg gat gac ttg atg gga tcc aga agc aac ttt 816 Val Cys Glu Ile Cys Thr Glu Glu Leu Met Gly Ser Arg Ser Asn Phe 260 265 270 gat tcc act cta ata agc cca aac tct gtc ttt tct caa tgg cga gtg 864 Glu Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 275 280 285 gtc tgt gac tcc ttg gaa gat tat gat acc ctg gga aca ctt tgt aac 912 Val Cys Glu Ser Leu Gln Glu Tyr Glu Thr Leu Gly Thr Leu Cys Asn 290 295 300 agc acc gag gat ggg cca att agg aga aat cca gct gga aat gtg gcc 960 Ser Thr Gln Glu Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 305 310 315 320 aga cca atg gtg caa cgt ctt cct gaa cca cag gat gtc gct cag tgc 1008 Arg Pro Met Val Gln Arg Leu Pro Gln Pro Gln Glu Val Ala Gln Cys 325 330 335 ttg gaa gtt ggt tta ttt gac acg cct cct ttt tat tcc aac tct aca 1056 Leu Gln Val Gly Leu Phe Glu Thr Pro Pro Phe Tyr Ser Asn Ser Thr 340 345 350 aac agt ttc cga aac aca gtg gaa ggt tac agt gac ccc acg gga aag 1104 Asn Ser Phe Arg Asn Thr Val Gln Gly Tyr Ser Glu Pro Thr Gly Lys 355 360 365 tat gac cct gct gtt cga agt ctt cac aat ttg gct cat cta ttc ctg 1152 Tyr Glu Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu 370 375 380 aat gga aca ggg gga caa acc cat ttg tct cca aat gat cct att ttt 1200 Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Glu Pro Ile Phe 385 390 395 400 gtc ctc ctg cac acc ttc aca gat gca gtc ttt gat gaa tgg ctg agg 1248 Val Leu Leu His Thr Phe Thr Glu Ala Val Phe Glu Gln Trp Leu Arg 405 410 415 aga tac aat gct gat ata tcc aca ttt cca ttg gaa aat gcc cct att 1296 Arg Tyr Asn Ala Glu Ile Ser Thr Phe Pro Leu Gln Asn Ala Pro Ile 420 425 430 gga cat aat aga caa tac aac atg gtg cca ttc tgg ccc cca gtc acc 1344 Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 435 440 445 aac aca gaa atg ttt gtt act gct cca gac aac ctg gga tac act tat 1392 Asn Thr Gln Met Phe Val Thr Ala Pro Glu Asn Leu Gly Tyr Thr Tyr 450 455 460 gaa att caa tgg cca agt cgg gag ttt agt gta cct gag cag ttc ccc 1440 Gln Ile Gln Trp Pro Ser Arg Gln Phe Ser Val Pro Gln Gln Phe Pro 465 470 475 480 cga gtc tgc atg acg gtg gac agc cta gtg aac aag gag tgc tgc cca 1488 Arg Val Cys Met Thr Val Glu Ser Leu Val Asn Lys Gln Cys Cys Pro 485 490 495 cgc ctg ggt gca gag tcg gcc aat gtc tgt ggc tct cag caa ggc cgg 1536 Arg Leu Gly Ala Gln Ser Ala Asn Val Cys Gly Ser Gln Gln Gly Arg 500 505 510 ggg cag tgc aca gag gtg cga gcc gac aca agg ccc tgg agt ggt ccc 1584 Gly Gln Cys Thr Gln Val Arg Ala Glu Thr Arg Pro Trp Ser Gly Pro 515 520 525 tac atc cta cga aac cag gat gac cgt gag ctg tgg cca aga aaa ttc 1632 Tyr Ile Leu Arg Asn Gln Glu Glu Arg Gln Leu Trp Pro Arg Lys Phe 530 535 540 ttc cac cgg acc tgc aag tgc aca gga aac ttt gcc ggc tat aat tgt 1680 Phe His Arg Thr Cys Lys Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys 545 550 555 560 gga gac tgc aag ttt ggc tgg acc ggt ccc aac tgc gag cgg aag aaa 1728 Gly Glu Cys Lys Phe Gly Trp Thr Gly Pro Asn Cys Gln Arg Lys Lys 565 570 575 cca cca gtg att cgg cag aac atc cat tcc ttg agt cct cag gaa aga 1776 Pro Pro Val Ile Arg Gln Asn Ile His Ser Leu Ser Pro Gln Gln Arg 580 585 590 gag cag ttc ttg ggc gcc tta gat ctc gcg aag aag aga gta cac ccc 1824 Gln Gln Phe Leu Gly Ala Leu Glu Leu Ala Lys Lys Arg Val His Pro 595 600 605 gac tac gtg atc acc aca caa cac tgg ctg ggc ctg ctt ggg ccc aat 1872 Glu Tyr Val Ile Thr Thr Gln His Trp Leu Gly Leu Leu Gly Pro Asn 610 615 620 gga acc cag ccg cag ttt gcc aac tgc agt gtt tat gat ttt ttt gtg 1920 Gly Thr Gln Pro Gln Phe Ala Asn Cys Ser Val Tyr Glu Phe Phe Val 625 630 635 640 tgg ctc cat tat tat tct gtt aga gat aca tta tta gga cca gga cgc 1968 Trp Leu His Tyr Tyr Ser Val Arg Glu Thr Leu Leu Gly Pro Gly Arg 645 650 655 ccc tac agg gcc ata gat ttc tca cat caa gga cct gca ttt gtt acc 2016 Pro Tyr Arg Ala Ile Glu Phe Ser His Gln Gly Pro Ala Phe Val Thr 660 665 670 tgg cac cgg tac cat ttg ttg tgt ctg gaa aga gat ctc cag cga ctc 2064 Trp His Arg Tyr His Leu Leu Cys Leu Gln Arg Glu Leu Gln Arg Leu 675 680 685 att ggc aat gag tct ttt gct ttg ccc tac tgg aac ttt gcc act ggg 2112 Ile Gly Asn Gln Ser Phe Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly 690 695 700 agg aac gag tgt gat gtg tgt aca gac cag ctg ttt ggg gca gcg aga 2160 Arg Asn Gln Cys Glu Val Cys Thr Glu Gln Leu Phe Gly Ala Ala Arg 705 710 715 720 cca gac gat ccg act ctg att agt cgg aac tca aga ttc tcc agc tgg 2208 Pro Glu Glu Pro Thr Leu Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp 725 730 735 gaa act gtc tgt gat agc ttg gat gac tac aac cac ctg gtc acc ttg 2256 Gln Thr Val Cys Glu Ser Leu Glu Glu Tyr Asn His Leu Val Thr Leu 740 745 750 tgc aat gga acc tat gaa ggt ttg ctg aga aga aat caa atg gga aga 2304 Cys Asn Gly Thr Tyr Gln Gly Leu Leu Arg Arg Asn Gln Met Gly Arg 755 760 765 aac agc atg aaa ttg cca acc tta aaa gac ata cga gat tgc ctg tct 2352 Asn Ser Met Lys Leu Pro Thr Leu Lys Glu Ile Arg Glu Cys Leu Ser 770 775 780 ctc cag aag ttt gac aat cct ccc ttc ttc cag aac tct acc ttc agt 2400 Leu Gln Lys Phe Glu Asn Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser 785 790 795 800 ttc agg aat gct ttg gaa ggg ttt gat aaa gca gat ggg act ctg gat 2448 Phe Arg Asn Ala Leu Gln Gly Phe Glu Lys Ala Glu Gly Thr Leu Glu 805 810 815 tct caa gtg atg agc ctt cat aat ttg gtt cat tcc ttc ctg aac ggg 2496 Ser Gln Val Met Ser Leu His Asn Leu Val His Ser Phe Leu Asn Gly 820 825 830 aca aac gct ttg cca cat tca gcc gcc aat gat ccc att ttt gtg gtt 2544 Thr Asn Ala Leu Pro His Ser Ala Ala Asn Glu Pro Ile Phe Val Val 835 840 845 ctt cat tcc ttt act gat gcc atc ttt gat gag tgg atg aaa aga ttt 2592 Leu His Ser Phe Thr Glu Ala Ile Phe Glu Gln Trp Met Lys Arg Phe 850 855 860 aat cct cct gca gat gcc tgg cct cag gag ctg gcc cct att ggt cac 2640 Asn Pro Pro Ala Glu Ala Trp Pro Gln Gln Leu Ala Pro Ile Gly His 865 870 875 880 aat cgg atg tac aac atg gtt cct ttc ttc cct cca gtg act aat gaa 2688 Asn Arg Met Tyr Asn Met Val Pro Phe Phe Pro Pro Val Thr Asn Gln 885 890 895 gaa ctc ttt tta acc tca gac caa ctt ggc tac agc tat gcc atc gat 2736 Gln Leu Phe Leu Thr Ser Glu Gln Leu Gly Tyr Ser Tyr Ala Ile Glu 900 905 910 ctg cca gtt tca gtt gaa gaa act cca ggt tgg ccc aca act cat ttc 2784 Leu Pro Val Ser Val Gln Gln Thr Pro Gly Trp Pro Thr Thr His Phe 915 920 925 cct aga gcc tgt gtc tcc tct aag aac ctg atg gag aag gaa tgc tgt 2832 Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Gln Lys Gln Cys Cys 930 935 940 cca ccg tgg agc ggg gac agg agt ccc tgt ggc cag ctt tca ggc aga 2880 Pro Pro Trp Ser Gly Glu Arg Ser Pro Cys Gly Gln Leu Ser Gly Arg 945 950 955 960 ggt tcc tgt cag aat atc ctt ctg tcc aat gca cca ctt ggg cct caa 2928 Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln 965 970 975 ttt ccc ttc aca ggg gtg gat gac cgg gag tcg tgg cct tcc gtc ttt 2976 Phe Pro Phe Thr Gly Val Glu Glu Arg Gln Ser Trp Pro Ser Val Phe 980 985 990 tat aat agg acc tgc cag tgc tct ggc aac ttc atg gga ttc aac tgt 3024 Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met Gly Phe Asn Cys 995 1000 1005 gga aac tgc aag ttt ggc ttt tgg gga cca aac tgc aca gag aga cga 3072 Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys Thr Gln Arg Arg 1010 1015 1020 ctc ttg gtg aga aga aac atc ttc gat ttg agt gcc cca gag aag gac 3120 Leu Leu Val Arg Arg Asn Ile Phe Glu Leu Ser Ala Pro Gln Lys Glu 1025 1030 1035 1040 aaa ttt ttt gcc tac ctc act tta gca aag cat acc atc agc tca gac 3168 Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr Ile Ser Ser Glu 1045 1050 1055 tat gtc atc ccc ata ggg acc tat ggc caa atg aaa aat gga tca aca 3216 Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys Asn Gly Ser Thr 1060 1065 1070 ccc atg ttt aac gac atc aat att tat gac ctc ttt gtc tgg atg cat 3264 Pro Met Phe Asn Glu Ile Asn Ile Tyr Glu Leu Phe Val Trp Met His 1075 1080 1085 tat tat gtg tca atg gat gca ctg ctt ggg gga tct gaa atc tgg aga 3312 Tyr Tyr Val Ser Met Glu Ala Leu Leu Gly Gly Ser Gln Ile Trp Arg 1090 1095 1100 gac att gat ttt gcc cat gaa gca cca gct ttt ctg cct tgg cat aga 3360 Glu Ile Glu Phe Ala His Gln Ala Pro Ala Phe Leu Pro Trp His Arg 1105 1110 1115 1120 ctc ttc ttg ttg cgg tgg gaa caa gaa atc cag aag ctg aca gga gat 3408 Leu Phe Leu Leu Arg Trp Gln Gln Gln Ile Gln Lys Leu Thr Gly Glu 1125 1130 1135 gaa aac ttc act att cca tat tgg gac tgg cgg gat gca gaa aag tgt 3456 Gln Asn Phe Thr Ile Pro Tyr Trp Glu Trp Arg Glu Ala Gln Lys Cys 1140 1145 1150 gac att tgc aca gat gag tac atg gga ggt cag cac ccc aca aat cct 3504 Glu Ile Cys Thr Glu Gln Tyr Met Gly Gly Gln His Pro Thr Asn Pro 1155 1160 1165 aac tta ctc agc cca gca tca ttc ttc tcc tct tgg cag att gtc tgt 3552 Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp Gln Ile Val Cys 1170 1175 1180 agc cga ttg gag gag tac aac agc cat cag tct tta tgc aat gga acg 3600 Ser Arg Leu Gln Gln Tyr Asn Ser His Gln Ser Leu Cys Asn Gly Thr 1185 1190 1195 1200 ccc gag gga cct tta cgg cgt aat cct gga aac cat gac aaa tcc aga 3648 Pro Gln Gly Pro Leu Arg Arg Asn Pro Gly Asn His Glu Lys Ser Arg 1205 1210 1215 acc cca agg ctc ccc tct tca gct gat gta gaa ttt tgc ctg agt ttg 3696 Thr Pro Arg Leu Pro Ser Ser Ala Glu Val Gln Phe Cys Leu Ser Leu 1220 1225 1230 acc caa tat gaa tct ggt tcc atg gat aaa gct gcc aat ttc agc ttt 3744 Thr Gln Tyr Gln Ser Gly Ser Met Glu Lys Ala Ala Asn Phe Ser Phe 1235 1240 1245 aga aat aca ctg gaa gga ttt gct agt cca ctt act ggg ata gcg gat 3792 Arg Asn Thr Leu Gln Gly Phe Ala Ser Pro Leu Thr Gly Ile Ala Glu 1250 1255 1260 gcc tct caa agc agc atg cac aat gcc ttg cac atc tat atg aat gga 3840 Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile Tyr Met Asn Gly 1265 1270 1275 1280 aca atg tcc cag gta cag gga tct gcc aac gat cct atc ttc ctt ctt 3888 Thr Met Ser Gln Val Gln Gly Ser Ala Asn Glu Pro Ile Phe Leu Leu 1285 1290 1295 cac cat gca ttt gtt gac agt att ttt gag cag tgg ctc cga agg cac 3936 His His Ala Phe Val Glu Ser Ile Phe Gln Gln Trp Leu Arg Arg His 1300 1305 1310 cgt cct ctt caa gaa gtt tat cca gaa gcc aat gca ccc att gga cat 3984 Arg Pro Leu Gln Gln Val Tyr Pro Gln Ala Asn Ala Pro Ile Gly His 1315 1320 1325 aac cgg gaa tcc tac atg gtt cct ttt ata cca ctg tac aga aat ggt 4032 Asn Arg Gln Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr Arg Asn Gly 1330 1335 1340 gat ttc ttt att tca tcc aaa gat ctg ggc tat gac tat agc tat cta 4080 Glu Phe Phe Ile Ser Ser Lys Glu Leu Gly Tyr Glu Tyr Ser Tyr Leu 1345 1350 1355 1360 caa gat tca gac cca gac tct ttt caa gac tac att aag tcc tat ttg 4128 Gln Glu Ser Glu Pro Glu Ser Phe Gln Glu Tyr Ile Lys Ser Tyr Leu 1365 1370 1375 gaa caa gcg agt cgg atc tgg tca 4152 Gln Gln Ala Ser Arg Ile Trp Ser 1380 12 1384 PRT Artificial Sequence Tri-hybrid antigen 12 Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 5 10 15 Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 20 25 30 Val Gln Ala Leu Arg Ser Gly Met Cys Cys Pro Glu Leu Ser Pro Val 35 40 45 Ser Gly Pro Gly Thr Glu Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 50 55 60 Cys Gln Ala Val Thr Ala Glu Ser Arg Pro His Ser Pro Gln Tyr Pro 65 70 75 80 His Glu Gly Arg Glu Glu Arg Gln Val Trp Pro Leu Arg Phe Phe Asn 85 90 95 Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 100 105 110 Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Glu Gln Arg Val Leu Ile 115 120 125 Val Arg Arg Asn Leu Leu Glu Leu Ser Lys Gln Gln Lys Asn His Phe 130 135 140 Val Arg Ala Leu Glu Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 145 150 155 160 Ile Ala Thr Arg Arg Ser Gln Gln Ile Leu Gly Pro Glu Gly Asn Thr 165 170 175 Pro Gln Phe Gln Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 180 185 190 Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Gln Ser Phe 195 200 205 Gly Gln Val Glu Phe Ser His Gln Gly Pro Ala Phe Leu Thr Trp His 210 215 220 Arg Tyr His Leu Leu Arg Leu Gln Lys Glu Met Gln Gln Met Leu Gln 225 230 235 240 Gln Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 245 250 255 Val Cys Glu Ile Cys Thr Glu Glu Leu Met Gly Ser Arg Ser Asn Phe 260 265 270 Glu Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 275 280 285 Val Cys Glu Ser Leu Gln Glu Tyr Glu Thr Leu Gly Thr Leu Cys Asn 290 295 300 Ser Thr Gln Glu Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 305 310 315 320 Arg Pro Met Val Gln Arg Leu Pro Gln Pro Gln Glu Val Ala Gln Cys 325 330 335 Leu Gln Val Gly Leu Phe Glu Thr Pro Pro Phe Tyr Ser Asn Ser Thr 340 345 350 Asn Ser Phe Arg Asn Thr Val Gln Gly Tyr Ser Glu Pro Thr Gly Lys 355 360 365 Tyr Glu Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu 370 375 380 Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Glu Pro Ile Phe 385 390 395 400 Val Leu Leu His Thr Phe Thr Glu Ala Val Phe Glu Gln Trp Leu Arg 405 410 415 Arg Tyr Asn Ala Glu Ile Ser Thr Phe Pro Leu Gln Asn Ala Pro Ile 420 425 430 Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 435 440 445 Asn Thr Gln Met Phe Val Thr Ala Pro Glu Asn Leu Gly Tyr Thr Tyr 450 455 460 Gln Ile Gln Trp Pro Ser Arg Gln Phe Ser Val Pro Gln Gln Phe Pro 465 470 475 480 Arg Val Cys Met Thr Val Glu Ser Leu Val Asn Lys Gln Cys Cys Pro 485 490 495 Arg Leu Gly Ala Gln Ser Ala Asn Val Cys Gly Ser Gln Gln Gly Arg 500 505 510 Gly Gln Cys Thr Gln Val Arg Ala Glu Thr Arg Pro Trp Ser Gly Pro 515 520 525 Tyr Ile Leu Arg Asn Gln Glu Glu Arg Gln Leu Trp Pro Arg Lys Phe 530 535 540 Phe His Arg Thr Cys Lys Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys 545 550 555 560 Gly Glu Cys Lys Phe Gly Trp Thr Gly Pro Asn Cys Gln Arg Lys Lys 565 570 575 Pro Pro Val Ile Arg Gln Asn Ile His Ser Leu Ser Pro Gln Gln Arg 580 585 590 Gln Gln Phe Leu Gly Ala Leu Glu Leu Ala Lys Lys Arg Val His Pro 595 600 605 Glu Tyr Val Ile Thr Thr Gln His Trp Leu Gly Leu Leu Gly Pro Asn 610 615 620 Gly Thr Gln Pro Gln Phe Ala Asn Cys Ser Val Tyr Glu Phe Phe Val 625 630 635 640 Trp Leu His Tyr Tyr Ser Val Arg Glu Thr Leu Leu Gly Pro Gly Arg 645 650 655 Pro Tyr Arg Ala Ile Glu Phe Ser His Gln Gly Pro Ala Phe Val Thr 660 665 670 Trp His Arg Tyr His Leu Leu Cys Leu Gln Arg Glu Leu Gln Arg Leu 675 680 685 Ile Gly Asn Gln Ser Phe Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly 690 695 700 Arg Asn Gln Cys Glu Val Cys Thr Glu Gln Leu Phe Gly Ala Ala Arg 705 710 715 720 Pro Glu Glu Pro Thr Leu Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp 725 730 735 Gln Thr Val Cys Glu Ser Leu Glu Glu Tyr Asn His Leu Val Thr Leu 740 745 750 Cys Asn Gly Thr Tyr Gln Gly Leu Leu Arg Arg Asn Gln Met Gly Arg 755 760 765 Asn Ser Met Lys Leu Pro Thr Leu Lys Glu Ile Arg Glu Cys Leu Ser 770 775 780 Leu Gln Lys Phe Glu Asn Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser 785 790 795 800 Phe Arg Asn Ala Leu Gln Gly Phe Glu Lys Ala Glu Gly Thr Leu Glu 805 810 815 Ser Gln Val Met Ser Leu His Asn Leu Val His Ser Phe Leu Asn Gly 820 825 830 Thr Asn Ala Leu Pro His Ser Ala Ala Asn Glu Pro Ile Phe Val Val 835 840 845 Leu His Ser Phe Thr Glu Ala Ile Phe Glu Gln Trp Met Lys Arg Phe 850 855 860 Asn Pro Pro Ala Glu Ala Trp Pro Gln Gln Leu Ala Pro Ile Gly His 865 870 875 880 Asn Arg Met Tyr Asn Met Val Pro Phe Phe Pro Pro Val Thr Asn Gln 885 890 895 Gln Leu Phe Leu Thr Ser Glu Gln Leu Gly Tyr Ser Tyr Ala Ile Glu 900 905 910 Leu Pro Val Ser Val Gln Gln Thr Pro Gly Trp Pro Thr Thr His Phe 915 920 925 Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Gln Lys Gln Cys Cys 930 935 940 Pro Pro Trp Ser Gly Glu Arg Ser Pro Cys Gly Gln Leu Ser Gly Arg 945 950 955 960 Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln 965 970 975 Phe Pro Phe Thr Gly Val Glu Glu Arg Gln Ser Trp Pro Ser Val Phe 980 985 990 Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met Gly Phe Asn Cys 995 1000 1005 Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys Thr Gln Arg Arg 1010 1015 1020 Leu Leu Val Arg Arg Asn Ile Phe Glu Leu Ser Ala Pro Gln Lys Glu 1025 1030 1035 1040 Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr Ile Ser Ser Glu 1045 1050 1055 Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys Asn Gly Ser Thr 1060 1065 1070 Pro Met Phe Asn Glu Ile Asn Ile Tyr Glu Leu Phe Val Trp Met His 1075 1080 1085 Tyr Tyr Val Ser Met Glu Ala Leu Leu Gly Gly Ser Gln Ile Trp Arg 1090 1095 1100 Glu Ile Glu Phe Ala His Gln Ala Pro Ala Phe Leu Pro Trp His Arg 1105 1110 1115 1120 Leu Phe Leu Leu Arg Trp Gln Gln Gln Ile Gln Lys Leu Thr Gly Glu 1125 1130 1135 Gln Asn Phe Thr Ile Pro Tyr Trp Glu Trp Arg Glu Ala Gln Lys Cys 1140 1145 1150 Glu Ile Cys Thr Glu Gln Tyr Met Gly Gly Gln His Pro Thr Asn Pro 1155 1160 1165 Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp Gln Ile Val Cys 1170 1175 1180 Ser Arg Leu Gln Gln Tyr Asn Ser His Gln Ser Leu Cys Asn Gly Thr 1185 1190 1195 1200 Pro Gln Gly Pro Leu Arg Arg Asn Pro Gly Asn His Glu Lys Ser Arg 1205 1210 1215 Thr Pro Arg Leu Pro Ser Ser Ala Glu Val Gln Phe Cys Leu Ser Leu 1220 1225 1230 Thr Gln Tyr Gln Ser Gly Ser Met Glu Lys Ala Ala Asn Phe Ser Phe 1235 1240 1245 Arg Asn Thr Leu Gln Gly Phe Ala Ser Pro Leu Thr Gly Ile Ala Glu 1250 1255 1260 Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile Tyr Met Asn Gly 1265 1270 1275 1280 Thr Met Ser Gln Val Gln Gly Ser Ala Asn Glu Pro Ile Phe Leu Leu 1285 1290 1295 His His Ala Phe Val Glu Ser Ile Phe Gln Gln Trp Leu Arg Arg His 1300 1305 1310 Arg Pro Leu Gln Gln Val Tyr Pro Gln Ala Asn Ala Pro Ile Gly His 1315 1320 1325 Asn Arg Gln Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr Arg Asn Gly 1330 1335 1340 Glu Phe Phe Ile Ser Ser Lys Glu Leu Gly Tyr Glu Tyr Ser Tyr Leu 1345 1350 1355 1360 Gln Glu Ser Glu Pro Glu Ser Phe Gln Glu Tyr Ile Lys Ser Tyr Leu 1365 1370 1375 Gln Gln Ala Ser Arg Ile Trp Ser 1380 13 529 PRT human Tyrosinase (GenBank Accession No. NP_000363) 13 Met Leu Leu Ala Val Leu Tyr Cys Leu Leu Trp Ser Phe Gln Thr Ser 5 10 15 Ala Gly His Phe Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Glu 20 25 30 Lys Glu Cys Cys Pro Pro Trp Ser Gly Asp Arg Ser Pro Cys Gly Gln 35 40 45 Leu Ser Gly Arg Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro 50 55 60 Leu Gly Pro Gln Phe Pro Phe Thr Gly Val Asp Asp Arg Glu Ser Trp 65 70 75 80 Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met 85 90 95 Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys 100 105 110 Thr Glu Arg Arg Leu Leu Val Arg Arg Asn Ile Phe Asp Leu Ser Ala 115 120 125 Pro Glu Lys Asp Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr 130 135 140 Ile Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys 145 150 155 160 Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Ile Tyr Asp Leu Phe 165 170 175 Val Trp Met His Tyr Tyr Val Ser Met Asp Ala Leu Leu Gly Gly Ser 180 185 190 Glu Ile Trp Arg Asp Ile Asp Phe Ala His Glu Ala Pro Ala Phe Leu 195 200 205 Pro Trp His Arg Leu Phe Leu Leu Arg Trp Glu Gln Glu Ile Gln Lys 210 215 220 Leu Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr Trp Asp Trp Arg Asp 225 230 235 240 Ala Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr Met Gly Gly Gln His 245 250 255 Pro Thr Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp 260 265 270 Gln Ile Val Cys Ser Arg Leu Glu Glu Tyr Asn Ser His Gln Ser Leu 275 280 285 Cys Asn Gly Thr Pro Glu Gly Pro Leu Arg Arg Asn Pro Gly Asn His 290 295 300 Asp Lys Ser Arg Thr Pro Arg Leu Pro Ser Ser Ala Asp Val Glu Phe 305 310 315 320 Cys Leu Ser Leu Thr Gln Tyr Glu Ser Gly Ser Met Asp Lys Ala Ala 325 330 335 Asn Phe Ser Phe Arg Asn Thr Leu Glu Gly Phe Ala Ser Pro Leu Thr 340 345 350 Gly Ile Ala Asp Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile 355 360 365 Tyr Met Asn Gly Thr Met Ser Gln Val Gln Gly Ser Ala Asn Asp Pro 370 375 380 Ile Phe Leu Leu His His Ala Phe Val Asp Ser Ile Phe Glu Gln Trp 385 390 395 400 Leu Arg Arg His Arg Pro Leu Gln Glu Val Tyr Pro Glu Ala Asn Ala 405 410 415 Pro Ile Gly His Asn Arg Glu Ser Tyr Met Val Pro Phe Ile Pro Leu 420 425 430 Tyr Arg Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp 435 440 445 Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr Ile 450 455 460 Lys Ser Tyr Leu Glu Gln Ala Ser Arg Ile Trp Ser Trp Leu Leu Gly 465 470 475 480 Ala Ala Met Val Gly Ala Val Leu Thr Ala Leu Leu Ala Gly Leu Val 485 490 495 Ser Leu Leu Cys Arg His Lys Arg Lys Gln Leu Pro Glu Glu Lys Gln 500 505 510 Pro Leu Leu Met Glu Lys Glu Asp Tyr His Ser Leu Tyr Gln Ser His 515 520 525 Leu 529 14 537 PRT human TRP-1 (GenBank Accession No. NP_000541) 14 Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 5 10 15 Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 20 25 30 Val Glu Ala Leu Arg Ser Gly Met Cys Cys Pro Asp Leu Ser Pro Val 35 40 45 Ser Gly Pro Gly Thr Asp Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 50 55 60 Cys Glu Ala Val Thr Ala Asp Ser Arg Pro His Ser Pro Gln Tyr Pro 65 70 75 80 His Asp Gly Arg Asp Asp Arg Glu Val Trp Pro Leu Arg Phe Phe Asn 85 90 95 Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 100 105 110 Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Asp Gln Arg Val Leu Ile 115 120 125 Val Arg Arg Asn Leu Leu Asp Leu Ser Lys Glu Glu Lys Asn His Phe 130 135 140 Val Arg Ala Leu Asp Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 145 150 155 160 Ile Ala Thr Arg Arg Ser Glu Glu Ile Leu Gly Pro Asp Gly Asn Thr 165 170 175 Pro Gln Phe Glu Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 180 185 190 Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Glu Ser Phe 195 200 205 Gly Glu Val Asp Phe Ser His Glu Gly Pro Ala Phe Leu Thr Trp His 210 215 220 Arg Tyr His Leu Leu Arg Leu Glu Lys Asp Met Gln Glu Met Leu Gln 225 230 235 240 Glu Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 245 250 255 Val Cys Asp Ile Cys Thr Asp Asp Leu Met Gly Ser Arg Ser Asn Phe 260 265 270 Asp Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 275 280 285 Val Cys Asp Ser Leu Glu Asp Tyr Asp Thr Leu Gly Thr Leu Cys Asn 290 295 300 Ser Thr Glu Asp Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 305 310 315 320 Arg Pro Met Val Gln Arg Leu Pro Glu Pro Gln Asp Val Ala Gln Cys 325 330 335 Leu Glu Val Gly Leu Phe Asp Thr Pro Pro Phe Tyr Ser Asn Ser Thr 340 345 350 Asn Ser Phe Arg Asn Thr Val Glu Gly Tyr Ser Asp Pro Thr Gly Lys 355 360 365 Tyr Asp Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu 370 375 380 Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Asp Pro Ile Phe 385 390 395 400 Val Leu Leu His Thr Phe Thr Asp Ala Val Phe Asp Glu Trp Leu Arg 405 410 415 Arg Tyr Asn Ala Asp Ile Ser Thr Phe Pro Leu Glu Asn Ala Pro Ile 420 425 430 Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 435 440 445 Asn Thr Glu Met Phe Val Thr Ala Pro Asp Asn Leu Gly Tyr Thr Tyr 450 455 460 Glu Ile Gln Trp Pro Ser Arg Glu Phe Ser Val Pro Glu Ile Ile Ala 465 470 475 480 Ile Ala Val Val Gly Ala Leu Leu Leu Val Ala Leu Ile Phe Gly Thr 485 490 495 Ala Ser Tyr Leu Ile Arg Ala Arg Arg Ser Met Asp Glu Ala Asn Gln 500 505 510 Pro Leu Leu Thr Asp Gln Tyr Gln Cys Tyr Ala Glu Glu Tyr Glu Lys 515 520 525 Leu Gln Asn Pro Asn Gln Ser Val Val 530 535 15 519 PRT human TRP-2(GenBank Accession No. NP_001913) 15 Met Ser Pro Leu Trp Trp Gly Phe Leu Leu Ser Cys Leu Gly Cys Lys 5 10 15 Ile Leu Pro Gly Ala Gln Gly Gln Phe Pro Arg Val Cys Met Thr Val 20 25 30 Asp Ser Leu Val Asn Lys Glu Cys Cys Pro Arg Leu Gly Ala Glu Ser 35 40 45 Ala Asn Val Cys Gly Ser Gln Gln Gly Arg Gly Gln Cys Thr Glu Val 50 55 60 Arg Ala Asp Thr Arg Pro Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln 65 70 75 80 Asp Asp Arg Glu Leu Trp Pro Arg Lys Phe Phe His Arg Thr Cys Lys 85 90 95 Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Asp Cys Lys Phe Gly 100 105 110 Trp Thr Gly Pro Asn Cys Glu Arg Lys Lys Pro Pro Val Ile Arg Gln 115 120 125 Asn Ile His Ser Leu Ser Pro Gln Glu Arg Glu Gln Phe Leu Gly Ala 130 135 140 Leu Asp Leu Ala Lys Lys Arg Val His Pro Asp Tyr Val Ile Thr Thr 145 150 155 160 Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro Gln Phe 165 170 175 Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr Tyr Ser 180 185 190 Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Arg Ala Ile Asp 195 200 205 Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His Arg Tyr His Leu 210 215 220 Leu Cys Leu Glu Arg Asp Leu Gln Arg Leu Ile Gly Asn Glu Ser Phe 225 230 235 240 Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly Arg Asn Glu Cys Asp Val 245 250 255 Cys Thr Asp Gln Leu Phe Gly Ala Ala Arg Pro Asp Asp Pro Thr Leu 260 265 270 Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp Glu Thr Val Cys Asp Ser 275 280 285 Leu Asp Asp Tyr Asn His Leu Val Thr Leu Cys Asn Gly Thr Tyr Glu 290 295 300 Gly Leu Leu Arg Arg Asn Gln Met Gly Arg Asn Ser Met Lys Leu Pro 305 310 315 320 Thr Leu Lys Asp Ile Arg Asp Cys Leu Ser Leu Gln Lys Phe Asp Asn 325 330 335 Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser Phe Arg Asn Ala Leu Glu 340 345 350 Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met Ser Leu 355 360 365 His Asn Leu Val His Ser Phe Leu Asn Gly Thr Asn Ala Leu Pro His 370 375 380 Ser Ala Ala Asn Asp Pro Ile Phe Val Val Leu His Ser Phe Thr Asp 385 390 395 400 Ala Ile Phe Asp Glu Trp Met Lys Arg Phe Asn Pro Pro Ala Asp Ala 405 410 415 Trp Pro Gln Glu Leu Ala Pro Ile Gly His Asn Arg Met Tyr Asn Met 420 425 430 Val Pro Phe Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu Thr Ser 435 440 445 Asp Gln Leu Gly Tyr Ser Tyr Ala Ile Asp Leu Pro Val Ser Val Glu 450 455 460 Glu Thr Pro Gly Trp Pro Thr Thr Leu Leu Val Val Met Gly Thr Leu 465 470 475 480 Val Ala Leu Val Gly Leu Phe Val Leu Leu Ala Phe Leu Gln Tyr Arg 485 490 495 Arg Leu Arg Lys Gly Tyr Thr Pro Leu Met Glu Thr His Leu Ser Ser 500 505 510 Lys Arg Tyr Thr Glu Glu Ala 515 16 42 DNA Artificial Sequence Amplification primer 16 gcggcggaat tccaattccc aagacagtgt gccactgttg ag 42 

We claim:
 1. An isolated DNA encoding a tri-hybrid melanoma antigen comprising tyrosinase or a fragment thereof, tyrosinase-related protein 1 (TRP-1) or a fragment thereof, and tyrosinase-related protein 2 (TRP-2) or a fragment thereof.
 2. The isolated DNA of claim 1, wherein the tri-hybrid melanoma antigen comprises SEQ ID NO:7 or a fragment thereof, SEQ ID NO:9 or a fragment thereof, or SEQ ID NO:11 or a fragment thereof.
 3. A cloning or expression vector comprising the DNA of claim 1 or
 2. 4. A host cell comprising the cloning or expression vector of claim
 3. 5. An isolated tri-hybrid melanoma antigen comprising tyrosinase or a fragment thereof, tyrosinase-related protein 1 (TRP-1) or a fragment thereof, and tyrosinase-related protein 2 (TRP-2) or a fragment thereof.
 6. The isolated tri-hybrid melanoma antigen of claim 5, wherein the tri-hybrid melanoma antigen comprises SEQ ID NO:8 or a fragment thereof, SEQ ID NO:10 or a fragment thereof, or SEQ ID NO:12 or a fragment thereof.
 7. A composition comprising the isolated DNA of claim 1 or 2 and a pharmaceutically acceptable carrier.
 8. A composition comprising the isolated tri-hybrid melanoma antigen of claim 5 or 6 and a pharmaceutically acceptable carrier.
 9. A composition for inhibiting melanosomal activity in an animal comprising a tri-hybrid melanoma antigen or a fragment thereof and a pharmaceutically acceptable carrier.
 10. A composition for inhibiting tumor growth in an animal comprising a tri-hybrid melanoma antigen or a fragment thereof and a pharmaceutically acceptable carrier.
 11. A composition for vaccination comprising a tri-hybrid melanoma antigen or a fragment thereof and a pharmaceutically acceptable carrier.
 12. The composition of any of claims 7-11, wherein the tri-hybrid melanoma antigen or a fragment thereof comprises tyrosinase or a fragment thereof, tyrosinase-related protein 1 (TRP-1) or a fragment thereof, and tyrosinase-related protein 2 (TRP-2) or a fragment thereof.
 13. The composition of any of claims 7-12, wherein the tri-hybrid melanoma antigen or a fragment thereof comprises SEQ ID NO:8 or a fragment thereof, SEQ ID NO:10 or a fragment thereof, or SEQ ID NO:12 or a fragment thereof.
 14. A method of eliciting an immune response against a melanosomal antigen in an animal comprising administering to the animal an effective amount of a tri-hybrid melanoma antigen or a fragment thereof.
 15. A method of treating a tumor in an animal comprising administering to the animal an effective amount of a tri-hybrid melanoma antigen or a fragment thereof.
 16. A method of vaccination in an animal comprising administering to the animal an effective amount of a tri-hybrid melanoma antigen or a fragment thereof.
 17. The method of any of claims 14-16, wherein the tri-hybrid melanoma antigen or a fragment thereof comprises tyrosinase or a fragment thereof, tyrosinase-related protein 1 (TRP-1) or a fragment thereof, and tyrosinase-related protein 2 (TRP-2) or a fragment thereof.
 18. The method of any of claims 14-17, wherein the tri-hybrid melanoma antigen or a fragment thereof comprises SEQ ID NO:8 or a fragment thereof, SEQ ID NO:10 or a fragment thereof, or SEQ ID NO:12 or a fragment thereof.
 19. The method of any of claims 14-18, wherein the method induces production of an antibody that specifically binds a melanosomal antigen.
 20. The method of any of claims 14-19, wherein the method induces production of IFNγ.
 21. The method of any of claims 14-20, wherein the method induces production of CD8 T cells.
 22. The method of any of claims 14-21, wherein the method induces production of cytotoxic lymphocytes specific for a melanosomal antigen.
 23. The method of any of claims 14-22, wherein the method is used to treat a condition associated with excess melanosomal antigen activity in the animal.
 24. The method of claim 23, wherein the condition is a tumor.
 25. The method of claim 24, wherein the tumor is a malignant tumor.
 26. The method of claim 25, wherein the malignant tumor is a carcinoma, sarcoma, leukemia, or a lymphoma.
 27. The method of any of claims 24-26, wherein the method prevents metastasis of the tumor.
 28. The method of claim 23, wherein the condition is a pre-malignant lesion.
 29. The method of claim 28, wherein the pre-malignant lesion is a adenoma or dysplastic lesion.
 30. The method of any of claims 14-29, wherein the tri-hybrid melanoma antigen is administered in combination with an adjuvant.
 31. The method of any of claims 14-30, wherein the animal is a mammal.
 32. The method of claim 31, wherein the animal is a human. 